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High-grade serous ovarian carcinoma commonly arises from fallopian tube secretory epithelium and is characterized by a high level of chromosomal instability. To model the acquisition of aneuploidy during early carcinogenesis, chromosome missegregation was induced in immortalized tubal epithelial cells, which proved acutely detrimental to cellular fitness. The phenotype was characterized by accumulation of misfolded proteins, activation of the unfolded protein response (UPR), decreased protein synthesis, and enhanced vulnerability to proteasome inhibition. However, chromosome missegregation also resulted in heightened transformation potential, assessed by colony formation in soft agar. Ovarian cancer cells retained intrinsic sensitivity to proteasome inhibitors under adherent culture conditions, but acquired resistance as spheroids (recapitulating their native configuration in ascites) by downregulating protein synthesis via mTORC1 suppression. Loss of PTEN drove constitutive mTORC1 activity, enhanced proteotoxic stress, as evidenced by UPR induction, and resensitized tumor spheroids to proteasome inhibition both in vitro and in vivo . In cohorts of primary ovarian carcinomas, mTORC1 and UPR signaling pathways were closely associated. These results implicate attenuation of protein synthesis as a protective mechanism in tumor spheroids, which may explain the overall poor response to bortezomib in clinical trials of patients with advanced ovarian cancer. However, patients with PTEN-deficient tumors may represent a subpopulation potentially amenable to treatment with proteasome inhibitors or other therapeutic agents that disrupt protein homeostasis. SIGNIFICANCE: Chromosome instability and protein synthesis are important factors that determine the efficacy of proteotoxic stress-inducing agents, such as proteasome inhibitors, in the treatment of ovarian cancer. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/21/5536/F1.large.jpg., (©2019 American Association for Cancer Research.)

The prolyl isomerase cyclophilin A (CypA) regulates the Jak2/Stat5 pathway, which is necessary for mammary differentiation and the pathogenesis of breast cancer. In this study, we assessed the role of this isomerase during mammary gland development and erbB2-driven tumorigenesis. Genetic deletion of CypA resulted in delayed mammary gland morphogenesis and differentiation with corresponding decrease in Jak2/Stat5 activation; mammary gland cross-transplantation confirmed this defect was epithelial in nature. Analysis of mammary stem and progenitor populations revealed significant disruption of epithelial maturation. Loss of CypA in the erbB2 transgenic mouse model revealed a marked increase in mammary tumor latency that correlated with decreased Stat5 activation, associated gene expression, and reduced epithelial cell proliferation. These results demonstrate an important role for CypA in the regulation of Jak2/Stat5-mediated biology in mammary epithelium, identifying this isomerase as a novel target for therapeutic intervention. Significance: These findings reveal cyclophilin A functions in normal mammary epithelial development and ErbB2-driven mammary tumorigenesis and suggest therapies targeting cyclophilin A may be efficacious for breast cancer treatment. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3877/F1.large.jpg Cancer Res; 78(14); 3877-87. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Src family kinases (SFK) integrate signal transduction for multiple receptors, regulating cellular proliferation, invasion, and metastasis in human cancer. Although Src is rarely mutated in human prostate cancer, SFK activity is increased in the majority of human prostate cancers. To determine the molecular mechanisms governing prostate cancer bone metastasis, FVB murine prostate epithelium was transduced with oncogenic v-Src. The prostate cancer cell lines metastasized in FVB mice to brain and bone. Gene expression profiling of the tumors identified activation of a CCR5 signaling module when the prostate epithelial cell lines were grown in vivo versus tissue cultures. The whole body, bone, and brain metastatic prostate cancer burden was reduced by oral CCR5 antagonist. Clinical trials of CCR5 inhibitors may warrant consideration in patients with CCR5 activation in their tumors., (©2014 American Association for Cancer Research.)

The transcription factor PPAR-γ plays various roles in lipid metabolism, inflammation, cellular differentiation, and apoptosis. PPAR-γ agonists used to treat diabetes may have utility in cancer treatment. Efatutazone is a novel later generation PPAR-γ agonist that selectively activates PPAR-γ target genes and has antiproliferative effects in a range of malignancies. In this study, we investigated PPAR-γ status in esophageal squamous cell carcinoma (ESCC) and investigated the antiproliferative effects of efatutazone. PPAR-γ was expressed heterogeneously in ESCC, in which it exhibited an inverse relationship with Ki-67 expression. PPAR-γ expression was associated independently with good prognosis in ESCC. Efatutazone, but not the conventional PPAR-γ agonist troglitazone, inhibited ESCC cell proliferation in vitro and in vivo. Mechanistic investigations suggested that efatutazone acted by upregulating p21Cip1 protein in the nucleus through inactivation of the Akt pathway and dephosphorylation of p21Cip1 at Thr145 without affecting the transcriptional activity of p21Cip1. We also found that treatment with efatutazone led to phosphorylation of the EGF receptor and activation of the mitogen-activated protein kinase (MAPK) pathway. Accordingly, the combination of efatutazone with the antiepithelial growth factor receptor antibody cetuximab synergized to negatively regulate the phosphoinositide 3-kinase-Akt and MAPK pathways. Together, our results suggest that efatutazone, alone or in combination with cetuximab, may offer therapeutic effects in ESCC.

Inhibitors of 5alpha-reductase (SRD5A) that lower intraprostatic levels of dihydrotestosterone (DHT) reduce the overall incidence of prostate cancer (PCa), but there is significant variation in chemopreventive activity between individual men. In seeking molecular alterations that might underlie this variation, we compared gene expression patterns in patients with localized PCa who were randomized to prostatectomy alone versus treatment with two different doses of the SRD5A inhibitor dutasteride. Prostatic levels of DHT were decreased by >90% in both dutasteride-treated patient groups versus the untreated patient group. Despite significant and uniform suppression of tissue DHT, unsupervised clustering based on prostatic gene expression did not discriminate these groups. However, subjects could be resolved into distinct cohorts characterized by high or low expression of genes regulated by the androgen receptor (AR), based solely on AR transcript expression. The higher-dose dutasteride treatment group was found to include significantly fewer cancers with TMPRSS2-ERG genetic fusions. Dutasteride treatment was associated with highly variable alterations in benign epithelial gene expression. Segregating subjects based on expression of AR and androgen-regulated genes revealed that patients are differentially sensitive to SRD5A inhibition. Our findings suggest that AR levels may predict the chemopreventive efficacy of SRD5A inhibitors.

The precise role of transforming growth factor (TGF)-beta signaling in head and neck squamous cell carcinoma (SCC) is not yet fully understood. Here, we report generation of an inducible head- and neck-specific knockout mouse model by crossing TGF-beta receptor I (Tgfbr1) floxed mice with K14-CreER(tam) mice. By applying tamoxifen to oral cavity of the mouse to induce Cre expression, we were able to conditionally delete Tgfbr1 in the mouse head and neck epithelia. On tumor induction with 7,12-dimethylbenz(a)anthracene (DMBA), 45% of Tgfbr1 conditional knockout (cKO) mice (n = 42) developed SCCs in the head and neck area starting from 16 weeks after treatment. However, no tumors were observed in the control littermates. A molecular analysis revealed an enhanced proliferation and loss of apoptosis in the basal layer of the head and neck epithelia of Tgfbr1 cKO mice 4 weeks after tamoxifen and DMBA treatment. The most notable finding of our study is that the phosphoinositide 3-kinase (PI3K)/Akt pathway was activated in SCCs that developed in the Tgfbr1 cKO mice on inactivation of TGF-beta signaling through Smad2/3 and DMBA treatment. These observations suggest that activation of Smad-independent pathways may contribute cooperatively with inactivation of Smad-dependent pathways to promote head and neck carcinogenesis in these mice. Our results revealed the critical role of the TGF-beta signaling pathway and its cross-talk with the PI3K/Akt pathway in suppressing head and neck carcinogenesis.

Mesothelioma is a unique and insidious tumor associated historically with occupational exposure to asbestos. The transcription factor, activator protein-1 (AP-1) is a major target of asbestos-induced signaling pathways. Here, we demonstrate that asbestos-induced mesothelial cell transformation is linked to increases in AP-1 DNA binding complexes and the AP-1 component, Fra-1. AP-1 binding to DNA was increased dramatically in mesothelioma cell lines in comparison to isolated rat pleural mesothelial (RPM) cells. Elevated levels of AP-1 complexes, including significant increases in c-Jun, JunB and Fra-1, were found in asbestos-exposed RPM cells, but only Fra-1 expression was significantly increased and protracted in both asbestos-exposed RPM cells and mesothelioma cell lines. Asbestos-induced Fra-1 expression in RPM cells was dependent on stimulation of the extracellular signal-regulated kinases (ERKs 1/2). Inhibition of ERK phosphorylation or transfection with dominant-negative fra-1 constructs reversed the transformed phenotype of mesothelioma cells and anchorage-independent growth in soft agar. In summary, we demonstrate that ERK-dependent Fra-1 is elevated in AP-1 complexes in response to asbestos fibers and is critical to the transformation of mesothelial cells.

The retinoblastoma (Rb) gene product is a prototypic tumor suppressor. Mice lacking the Rb gene are not viable and die in utero at approximately 13 days of gestation. In this study, we have rescued Rb-/- prostates by grafting pelvic organ rudiments from Rb-/- mouse embryos under the renal capsule of adult male nude mouse hosts. Grafts of embryonic pelvic organs developed into functional prostatic tissue. Some of the prostatic tissue generated was further used to construct chimeric prostatic tissue recombinants by combining wild-type rat urogenital mesenchyme (rUGM) with Rb-/- and Rb+/+ prostatic epithelium (PRE). The tissue recombinants were grown as subcapsular renal grafts and treated from the time of grafting with Silastic capsules containing 25 mg of testosterone plus 2.5 mg of estradiol. During 5-8 weeks of hormone treatment, rUGM+Rb+/+PRE tissue recombinants developed prostatic hyperplasia, whereas PRE in rUGM+Rb-/-PRE tissue recombinants developed hyperplasia, atypical hyperplasia, and carcinoma. During carcinogenesis in rUGM+Rb-/-PRE tissue recombinants, prostatic epithelial cells of the basal lineage disappeared, whereas the luminal cells underwent carcinogenesis. Epithelial E-cadherin almost totally disappeared. In all cases, epithelial PCNA labeling was elevated in tissue recombinants containing Rb-/- versus Rb+/+ epithelium. These epithelial changes were associated with almost total loss of smooth muscle cells in the stroma. In contrast, in untreated hosts rUGM+Rb+/+PRE tissue recombinants developed normally, and rUGM+Rb-/-PRE tissue recombinants developed mild epithelial hyperplasia. The results of this study demonstrate that Rb-/- prostatic tissue can be rescued from embryonic lethal mice and used to test its susceptibility to hormonal carcinogenesis. Deletion of the Rb gene predisposes prostatic epithelium to hyperplasia and increases proliferative activity Susceptibility to hormonal carcinogenesis in response to exogenous testosterone + estradiol is manifested in the progression from atypica hyperplasia to carcinoma. Thus, these findings demonstrate that the absence of the Rb tumor suppressor gene may predispose prostatic epithelial cells to carcinogenesis. Rescue of organs from Rb-/- embryos not only provides an opportunity to analyze the Rb gene pathway in the development and progression of prostate cancer but also provides an opportunity for specifically evaluating the role of the Rb pathway in development and carcinogenesis in other organs, such as the mammary gland and colon. Because rUGM greatly stimulates prostatic epithelial proliferation, the tissue recombinant model is a particularly useful tool for assessing the functional role of other genes in prostatic carcinogenesis through use of the appropriate transgenic or gene knockout mice.

The relationship between acute (<36 h) induction of apoptosis and longer-term (>72 h) intestinal histopathology was systematically investigated in vivo using p53 wild-type (+/+) and null (-/-) mice. Administration of the enterotoxin 5-fluorouracil (5-FU) at either 40 or 400 mg/kg to BDF1 mice induced an acute p53-dependent apoptosis in the crypts of both small intestine and midcolon. Although the amount of apoptosis was of the same order of magnitude at its peak (24 h) at both doses, only 400 mg/kg 5-FU brought about histopathological changes to the gut after 96 h, quantified as losses of crypt and villus cellularity. Only after the administration of 400 mg/kg 5-FU were mitotic index and DNA synthesis significantly suppressed in both small intestinal and midcolonic crypts at 24 h. This correlated with a prolonged, p53-dependent expression of p21waf-1/cip1. In p53 null (-/-) mice, significant reductions in both 5-FU-induced apoptosis and inhibition of cell cycle progression allowed retention of crypt integrity 96 h after 5-FU. These results show that quantitative measures of acute apoptosis in vivo may not accurately predict subsequent pathological changes in the gut. Rather, p53-dependent inhibition of cell cycle progression, together with cell loss by apoptosis, caused a loss of crypt integrity. Importantly, the tissue toxicity of 5-FU was genetically determined at a locus (p53) separate from that directly associated with drug action.

Protein kinase C activation leads to tight junctional leakiness and, consequently, to increased transepithelial (paracellular) solute flux across epithelial barriers. This leakiness is shown here to result in as much as a 20-fold increase in the transepithelial flux of insulin. Using an epithelial/fibroblast coculture model, this transepithelially transported insulin is shown to be biologically active. The 3T3 fibroblasts situated on one side of the epithelial barrier exhibited increased insulin binding and resulting DNA synthesis when the epithelial junctions were made leaky to insulin on the opposite side of the epithelial barrier. The dramatically enhanced permeability of macromolecules across epithelial cell layers undergoing protein kinase C activation may play a significant role in epithelial cancer, immunology, and drug delivery.

The 1-methyl-1-nitrosourea-induced rat mammary tumor model system is well studied, reproducible, and widely used. We have investigated whether these tumors possess higher telomerase activity than normal mammary tissue. Using the telomeric repeat amplification protocol assay, we found significantly higher telomerase activity in 36 mammary carcinomas than in 72 mammary glands of virgin rats. The level of telomerase activity in virgin rats was unaffected by strain, age, stage of the estrous cycle, or ovariectomy. However, mammary glands obtained from pregnant rats exhibited telomerase activity comparable to that found in the tumors, possibly reflecting the high epithelial content of these tissues. Indeed, isolated epithelial cells from virgin and pregnant mammary glands and from carcinomas had similar telomerase activities. Thus, telomerase activity is constitutive in the rat mammary epithelium and is not a unique characteristic of malignant transformation in this tissue. These results underscore the importance of attributing biochemical properties to specific cell types in a tissue, a situation not paralleled in the interpretation of data from in vitro models.

Previous research showed that treatment with conjugated linoleic acid (CLA) during the period of active mammary gland morphogenesis was sufficient to confer a lasting protection against subsequent mammary tumorigenesis induced by methylnitrosourea. The present study was designed to characterize certain morphological and biochemical changes of the mammary gland that might potentially render it less susceptible to cancer induction. Female Sprague Dawley rats were fed a 1% CLA diet from weaning until about 50 days of age. The mammary gland parameters under investigation included (a) the deposition of neutral lipid, (b) the identification and quantification of CLA and its metabolites, (c) the density of the epithelium, and (d) the proliferative activity of various structural components. Our results showed that CLA treatment did not affect total fat deposition in the mammary tissue nor the extent of epithelial invasion into the surrounding fat pad but was able to cause a 20% reduction in the density of the ductal-lobular tree as determined by digitized image analysis of the whole mounts. This was accompanied by a suppression of bromodeoxyuridine labeling in the terminal end buds and lobuloalveolar buds. The recovery of desaturation and elongation products of CLA in the mammary gland confirmed our prior suggestion that the metabolism of CLA might be critical to risk modulation. The significance of the above findings was investigated in a mammary carcinogenesis bioassay with the use of the dimethylbenz[a]anthracene model. When CLA was started at weaning and continued for 6 months until the end of the experiment, this schedule of supplementation produced essentially the same magnitude of mammary tumor inhibition in the dimethylbenz[a]anthracene model as that produced by 1 month of CLA feeding from weaning. The observation is consistent with the hypothesis that exposure to CLA during the time of mammary gland maturation may modify the developmental potential of a subset of target cells that are normally susceptible to carcinogen-induced transformation.

Asbestos and the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), increase c-fos and c-jun mRNA levels and AP-1 DNA binding activity in rat pleural mesothelial (RPM) cells, a target cell of asbestos-induced mesotheliomas (N. H. Heintz et al., Proc. Natl. Acad. Sci. USA, 90: 3299-3303, 1993). Because protein kinase C (PKC) is the intracellular receptor of phorbol ester tumor promoters and asbestos is a putative tumor promoter in the respiratory tract, we hypothesized that PKC might play a critical role in asbestos-induced cell signaling pathways associated with regulation of proto-oncogenes. Using a panel of PKC antibodies, we identified PKC alpha as the major PKC isozyme in RPM cells. We then pretreated cells with phorbol ester dibutyrate to down-modulate PKC or with calphostin C, a specific PKC inhibitor, to determine if depletion of PKC alpha could block asbestos-induced c-fos/c-jun expression. Quantitation of Northern blots showed that fiber-associated c-fos/c-jun mRNA levels were significantly lower either after PKC alpha down-modulation or pretreatment with calphostin C. In addition, to determine whether tyrosine kinases also were involved in proto-oncogene activation by asbestos, tyrphostin AG82 or herbimycin A was added to RPM cells before exposure to asbestos. These inhibitors decreased crocidolite-induced c-fos but not c-jun levels, suggesting that tyrosine kinases have different regulatory roles in asbestos-induced c-fos versus c-jun signaling pathways. The ability to block induction of asbestos-induced proto-oncogene expression using pharmacological intervention may be important in prevention and treatment of asbestos-induced proliferative diseases including lung cancers, mesothelioma, and pulmonary fibrosis.

Carrageenans are naturally occurring sulfated polysaccharides, widely used in commercial food preparation to improve the texture of processed foods. Because of their ubiquity in the diet and their observed preneoplastic effects in intestinal cells, their impact on human mammary myoepithelial cells in tissue culture was studied. At concentrations as low as 0.00014%, lambda-carrageenan was associated with disassembly of filaments with reduced immunostaining for vimentin, alpha-smooth muscle-specific actin, and gelsolin; increased staining for cytokeratin 14; and cell death. The absence of mammary myoepithelial cells is associated with invasive mammary malignancy; hence, the destruction of these cells in tissue culture by a low concentration of a widely used food additive suggests a dietary mechanism for mammary carcinogenesis not considered previously.

The Bcl-2 oncoprotein, which is expressed in a variety of human malignancies, blocks apoptosis induced by chemotherapeutic drugs, including the topoisomerase II inhibitor, etoposide. To determine the significance of Bcl-2 in etoposide-induced death of human epithelial tumor cells, HeLa S3 cells were transfected with human bcl-2 cDNA in the pSFFV expression vector, and stable Bcl-2-expressing clones established. In agreement with previous studies, Bcl-2 inhibited loss of cell viability (by trypan blue exclusion), the appearance of morphologically apoptotic cells, and the amount of low molecular weight DNA extracted after etoposide exposure (25 microns, 4 h). The degree of inhibition, compared to wild-type and vector control-transfected clones, differed according to the level of Bcl-2 protein expressed in the two clones studied. However, when cell survival was assessed by colony-forming assays, no significant differences were detected at any of the etoposide concentrations used. Although Bcl-2 inhibited etoposide-induced apoptosis, it had no effect on the formation of giant, multinucleated cells characteristic of mitotic catastrophe. Consequently, the ability of Bcl-2 to prevent apoptosis caused by chemotherapeutic drugs may not necessarily translate into increased survival of cells that express Bcl-2.

Multifunctional cytokines play important and only partially defined roles in mammary tumor development and progression. Normal human mammary epithelia] cells (MECs) constitutively produce interleukin (IL) 6, IL8, and a nonsecreted form of tumor necrosis factor. MEC transformation by different oncogenes is frequently associated with alterations of cytokine/growth factor production and responsiveness. This seems particularly true in the case of IL6. Histochemical studies showed that expression of immunoreactive IL6, as compared to normal tissue and to in situ lesions, is significantly reduced in invasive ductal carcinoma. Conversely, the expression of IL6 in invasive lobular carcinoma was enhanced. Expression of TGF-beta1 in mammary neoplasia was in general less intense than that seen in the normal mammary gland. In vitro studies partially supported the in vivo findings: expression of IL6 and TGF-beta1 was significantly down-regulated in cultures derived from both ductal carcinoma and peritumoral tissue. Similarly, responsiveness to IL6 and TGF-beta1 was significantly reduced in neoplastic MECs. The data suggest that alterations of cytokine pathways are present not only in mammary neoplasia, but also in pathologically unaffected breast tissues.

We investigated the mitochondrial toxicity of the lipophilic cation, MKT-077, and the role of mitochondria in selective malignant cell killing by this compound by examining the effect of MKT-077 on mitochondrial structure and function in treated cells and in isolated organelles. Results of this study demonstrate changes in mitochondrial ultrastructure that are induced by MKT-077 treatment in carcinoma cells but not in similarly treated normal epithelial cells. In addition, MKT-077 was found to inhibit respiratory activity in isolated intact mitochondria and electron transport activity in freeze-thawed mitochondrial membrane fragments in a dose-dependent manner. The concentration of MKT-077 necessary to obtain half-maximal inhibition of ADP-stimulated respiration was approximately 4-fold greater in mitochondria isolated from cells of the normal epithelial cell line, CV-1 (15 micrograms MKT-077/mg protein), as compared to the human colon carcinoma cell line, CX-1 (4 micrograms MKT-077/mg protein). Further, the data show a selective loss of mitochondrial DNA in CX-1 and CRL1420 cells (carcinoma) but not CV-1 cells (normal epithelial) treated with 3 microgram/ml MKT-077 for up to 3 days. Under the same conditions, nuclear DNA was unaffected in all three cell lines. The sensitivity of the cell lines tested to mitochondrial damage by MKT-077 correlates well with their sensitivity to cytotoxicity by MKT-077. These results demonstrate selective mitochondrial damage by MKT-077 at the cellular, biochemical, and molecular levels and suggest that selective effects on mitochondrial structure and function may provide a basis for the selective malignant cell killing exhibited by this compound.

The cell cycle inhibitor mimosine was used to examine the activation of the p34cdc2 protein kinase in S phase of the cell cycle. Addition of mimosine to cycling epithelial cells halted cell cycle traverse in S phase, coincident with an inhibition of p34cdc2 histone H1 kinase activity. Mimosine treatment did not alter p34cdc2 synthesis or turnover; however, overall phosphorylation of p34cdc2 was decreased to near undetectable levels. Although activity of p34cdc2 was inhibited, the ability of the protein to form high molecular weight complexes, a phenomenon associated with kinase activation in vivo, was not affected. These results indicate that p34cdc2 complex formation can occur in the absence of phosphorylation and that phosphorylation of p34cdc2 is then required to activate these preformed complexes.

We have introduced a human gastrin-releasing peptide receptor expression vector into an immortalized human bronchial epithelial cell normally unresponsive to the ligand bombesin. Successfully transfected cells express specific binding sites at a density similar to that found at the surface of human lung cancer cells and show an elevation of intracellular calcium concentration in response to bombesin. We found that cellular strains expressing the receptor showed a growth stimulation in response to bombesin in proportion to cell surface receptor density. We conclude that expression of bombesin receptors contributes to the growth potential of human bronchial epithelial cells.

Preferential retention and cytotoxicity of Rhodamine-123 (Rho-123) was originally reported in a number of carcinoma cell types isolated from a variety of tissues as compared to normal epithelial cells from a limited number of other tissues. In the present study, we have examined Rho-123 selectivity in normal and tumor cell lines isolated from the same tissue source, i.e., human breast. We found that: (a) in matched pairs of normal and carcinoma breast cells, Rho-123 displays no preferential retention in either cell type; (b) there is no preferential toxicity in carcinoma as compared to normal breast cells; in fact, one of the carcinoma cell lines (MDA-MB231) shows moderate resistance to this dye; (c) all of the human breast cell lines do not express P-glycoprotein-mediated multidrug resistance; (d) the normal monkey kidney epithelial cell line CV-1, which was originally used as a model to demonstrate the relative resistance of normal epithelial cells to this drug, is found to express high levels of the mdr-1 gene, is resistant to other multidrug-resistant drugs (taxol and vinblastine), and its resistance to Rho-123 as well as decreased Rho-123 retention can be reversed by verapamil; and (e) taxol and vinblastine are found to block increased Rho-123 efflux in CV-1 cells. Thus, overall the data suggest that preferential retention and cytotoxicity of Rho-123 in carcinoma versus normal epithelial cells is related to the differential expression of the mdr-1 gene.

Asbestos has been described as a physical carcinogen in that long thin fibers are generally more carcinogenic than shorter thicker ones. It has been hypothesized that long thin fibers disrupt chromosome behavior during mitosis, causing chromosome abnormalities which lead to cell transformation and neoplastic progression. Using high-resolution time lapse video-enhanced light microscopy and the uniquely suited lung epithelial cells of the newt Taricha granulosa, we have characterized for the first time the behavior of crocidolite asbestos fibers, and their interactions with chromosomes, during mitosis in living cells. We found that the keratin cage surrounding the mitotic spindle inhibited fiber migration, resulting in spindles with few fibers. As in interphase, fibers displayed microtubule-mediated saltatory movements. Fiber position was only slightly affected by the ejection forces of the spindle asters. Physical interactions between crocidolite fibers and chromosomes occurred randomly within the spindle and along its edge. Crocidolite fibers showed no affinity toward chromatin and most encounters ended with the fiber passively yielding to the chromosome. In a few encounters along the spindle edge the chromosome yielded to the fiber, which remained stationary as if anchored to the keratin cage. We suggest that fibers thin enough to be caught in the keratin cage and long enough to protrude into the spindle are those fibers with the ability to snag or block moving chromosomes.

3'-Azido-2',3'-dideoxythymidine (AZT, Retrovir, zidovudine), a nucleoside analogue currently used in the therapy of acquired immunodeficiency syndrome, induces papillomas and carcinomas in vaginal epithelium of mice as a result of lifetime drug administration. In this study, female CD-1 mice were administered AZT at doses of 180, 360, and 720 micrograms/ml of drinking water for 28 days to determine whether AZT became incorporated into vaginal DNA and whether this was associated with preneoplastic changes within the target tissue. In addition, bone marrow, a target for AZT-induced cytotoxicity in mice and humans, was examined for chromosomal aberrations. A positive correlation was observed between dose level of AZT, proliferation of cells in the basal layer of vaginal epithelium, and incorporation of AZT into vaginal DNA. Incorporation of AZT into vaginal DNA was originally detected by radioimmunoassay and confirmed by immunohistochemistry. An aberrant pattern for alpha 6 integrin distribution, similar to the pattern described in skin papillomas with high risk for malignant conversion, also increased with dose in mice given AZT. Chromosomal aberrations in bone marrow increased more than 4-fold in AZT-exposed animals. The genotoxicity demonstrated by incorporation of AZT into vaginal DNA and proliferation of vaginal epithelium may play an essential part in the ability of AZT to induce abnormal differentiation in vaginal epithelium and vaginal tumorigenesis in mice.

The neoplastic transformation of cultured rat liver epithelial cells by various means has consistently been associated with the development of resistance to the mito-inhibitory effect of transforming growth factor beta (TGF-beta), suggesting that such phenotype plays a mechanistic role during the transformation of these cells. We have studied the induction of the "TGF-beta-resistant" phenotype in a clonal strain of early passage WB-F344 normal cultured rat liver epithelial cells, the proliferation of which was markedly inhibited by TGF-beta. The control WB cells in continuous culture slowly developed TGF-beta resistance. However, when the same cells were exposed to step-wise increases of TGF-beta concentration in their culture medium, the development of TGF-beta resistance was accelerated. Cells which had been grown in medium containing 1 ng/ml TGF-beta developed colony-forming capacity in soft agar containing epidermal growth factor. Cells which were grown in media containing 5 and 10 ng/ml TGF-beta demonstrated a low level of colony-forming efficiency in soft agar medium without added epidermal growth factor and tumorigenicity in isogeneic rats. These TGF-beta-resistant cells also exhibited progressively increasing levels of expression of the c-fos and and myc mRNA, and increased resistance to the cytotoxicity of Adriamycin and melphalan. The latter phenomenon was accompanied by an increase in the mdr-1 mRNA expression, cellular glutathione level, and glutathione S-transferase activity. The results suggest that chronic exposure to high concentration of TGF-beta promotes the spontaneous neoplastic transformation of cultured rat liver epithelial cells, and that this process may represent one of the mechanisms of cellular adaptation for induction of the multidrug-resistant phenotype during the carcinogenesis of epithelial cells.

In the present study, we examine the effects of all-trans-retinoic acid (RA) and interferons-alpha and -gamma (IFN-alpha and IFN-gamma) on the growth of HPV16-immortalized cell lines, ECE16-1 and CaSki. Treating proliferating ECE16-1 cells with RA causes a concentration-dependent decrease in cell number. At 1 microM RA, cell growth is suppressed by 65% and the level of mRNA encoding cytokeratin K5, a biochemical marker of retinoid action, is also suppressed. In contrast, the level of transcript encoding the HPV16 oncogenes, E6 and E7, is reduced by only 5 to 10%. IFN-alpha at 1000 IU/ml or IFN-gamma at 200 IU/ml suppresses growth by 70%. This growth suppression by IFN-gamma is correlated with a > 90% reduction in E6/E7 mRNA levels. Additional growth suppression is observed upon simultaneous treatment with retinoid and interferon. Optimal suppression is observed in the presence of 200 IU/ml IFN-gamma and 1 microM RA. The rank order of effectiveness is IFN-gamma/RA > IFN-alpha/RA = IFN-gamma > RA > IFN-alpha. In contrast to the suppression of ECE16-1 cell growth, RA causes a concentration-dependent increase in CaSki cell number (50-60%) which is optimal at 1 microM RA. Cytokeratin K5 mRNA levels are markedly suppressed, and E6/E7 mRNA levels increased by 5% under these conditions. IFN-alpha at 1000 IU/ml or IFN-gamma at 200 IU/ml decreases CaSki cell growth by 20 and 45%, respectively, and 200 IU/ml of IFN-gamma reduce E6/E7 expression to undetectable levels. Addition of RA (1 microM) partially counters the IFN-dependent suppression of growth and E6/E7 mRNA levels. Our results suggest that retinoid-dependent changes in human papillomavirus-immortalized cervical cell proliferation are not always correlated with changes in E6/E7 transcript levels.

We detected low level expression of the gastrin-releasing peptide and neuromedin-B receptor mRNAs in cultures of human bronchial epithelium from 4 of 6 individuals. Bombesin receptor subtype-3 mRNA was undetectable in these cells. An elevation of intracellular calcium concentration was observed in response to bradykinin (6 of 6) and neurotensin (1 of 5) but not to bombesin (0 of 6), vasopressin (0 of 6), or cholecystokinin (0 of 3). In contrast, such responses are frequently noted in lung cancer cell lines. Bombesin did not stimulate the in vitro growth of an immortalized human bronchial epithelium cell line expressing low levels of bombesin receptor mRNAs. We conclude that bombesin receptors are expressed at low levels in human bronchial epithelium cells which may acquire greater responsiveness to multiple neuropeptides in the course of multistep carcinogenesis.

Certain lipophilic cations have been reported to display anticarcinoma activities because of their selective uptake and retention by mitochondria of cancer cells. Thus, these agents may comprise a unique class of agents directed against carcinoma. After screening more than 1000 lipophilic cations, we found that the monovalent lipophilic cation, 2,6-bis(4-amino-phenyl)-4-[4-(dimethylamino)phenyl]thiopyrylium chloride (AA1), displayed remarkable anticarcinoma activity both in vitro and in vivo. Unlike most other lipophilic cations, AA1 is stable and displays minimal light sensitivity. In vitro testing showed that AA1 was 10 times more toxic to the carcinoma cell line CX-1 than to the normal epithelial cell line CV-1. In vivo animal experiments showed that AA1 significantly prolonged the survival of mice implanted with tumors. For C57BL x DBA/2 F1 mice implanted with the mouse bladder carcinoma cell line, MB49, the treated:control ratio was 344%. For Swiss nu/nu mice implanted i.p. with the human melanoma cell line, LOX, the treated:control ratio was 341%. The most significant observation was obtained with Swiss nu/nu mice that were implanted i.p. with the human ovarian cell line, OVCAR-III. The treated:control ratio in this situation was greater than 450%. In all these tumor models, AA1 produced minimal toxicities. AA1 exhibited little inhibition of electron transport in isolated rat liver mitochondria; however, it inhibited mitochondrial ATPase with 50% inhibitory concentration of 6 microM. Compared with previously reported anticarcinoma lipophilic cations such as rhodamine 123 and dequalinium chloride, AA1 appeared to display more effective in vivo anticarcinoma activity. Thus, AA1 could be considered for further clinical development as a candidate for anticarcinoma chemotherapy.

Mucositis is a common, dose-limiting complication in patients receiving cancer chemotherapy, which appears to be a consequence of the rate of epithelial proliferation. The beta transforming growth factors have been shown to be negative regulators of epithelial cell proliferation. Here we show that transforming growth factor beta 3 administration reduced proliferation of oral epithelium in vitro and in vivo. Topical application of transforming growth factor beta 3 to the oral mucosa of the Syrian golden hamster prior to chemotherapy significantly reduced the incidence, severity, and duration of oral mucositis, reduced chemotherapy-associated weight loss, and increased survival.

Estrogen is essential for the growth of the normal mammary gland and most estrogen receptor (ER)-positive mammary carcinomas. To better understand the differences between the estrogen response pathways in normal and tumor cells, we have stably transfected ER-negative immortal, nontumorigenic human mammary epithelial cells and ER-negative breast cancer cells with an ER-encoding expression vector. Unexpectedly, estrogen treatment (1.0 nM) inhibited the proliferation of ER-transfected nontumorigenic and tumor-derived cells. The control transfectants and parental cells exhibited no response to estrogen concentrations as high as 1.0 microM. This inhibitory effect was attributed to a decreased growth rate and a perturbation of the cell cycle distribution by estrogen treatment of the ER transfectants. The inhibitory response was blocked by cotreatment with the antiestrogen ICI 164,384 as predicted for a pure antagonist of estrogen action. However, treatment with the antiestrogen hydroxytamoxifen caused growth inhibition, implying that hydroxytamoxifen acts as an agonist of estrogen action in ER-transfected cells. Since estrogen is a mitogenic and not a growth-inhibitory stimulus for ER-positive breast cancers and cell lines, we tested the effect of constitutive, high level expression of the ER in ER-positive tumor cells. Stable transfection of ER-positive MCF-7 and T47D cells with the ER expression vector yielded cells with varying amounts of ER. At ER levels comparable to those found in the ER-negative transfected cells, the MCF-7 and T47D ER transfectants were not inhibited by estrogen. These data suggest that ER-positive breast cancer cells can tolerate higher constitutive levels of ER expression than ER-negative cells. The mechanism by which this is accomplished may be an essential step in the process which yields ER-positive tumors.

Galactosyl beta-1,3-N-acetyl galactosamine (Gal beta-1,3-GalNAc) (Thomsen Friedenreich antigen), the Class I core sequence in O-linked oligosaccharide chains, behaves as an oncofetal antigen showing increased expression in many epithelial malignancies. Previous work has shown that peanut agglutinin (PNA), a lectin that binds Gal beta-1,3-GalNAc, stimulates proliferation in HT-29 (human colon cancer) cells and normal human colonic epithelium and this implies that cell surface glycoproteins which express Gal beta-1,3-GalNAc may play an important role in the regulation of epithelial cell proliferation. We have now studied the effect on epithelial cells of another dietary Gal beta-1,3-GalNAc-binding lectin, the edible mushroom Agaricus bisporus lectin (ABL). This differs from PNA in its ability to bind also to sialylated Gal beta-1,3-GalNAc. In contrast to PNA, ABL (25 micrograms/ml) inhibited incorporation of [3H]-thymidine into DNA of HT29 colon cancer cells by 87% (95% confidence limit, 85-89%), Caco-2 colon cancer cells by 16% (95% confidence limit, 12-20%), MCF-7 breast cancer cells by 50% (95% confidence limit, 47-52%), and Rama-27 rat mammary fibroblasts by 55% (95% confidence limit, 51-60%) when these cells were grown for 24 h in serum-free medium. When assessed by cell count, similar inhibition of proliferation of HT29 cells by ABL was found. In the presence of 2% fetal calf serum (which contains the ABL-binding glycoprotein fetuin), the inhibitory effect of ABL on cell proliferation was still demonstrable but at increased ABL concentration (60 micrograms/ml for 49% inhibition). Ten micrograms/ml ABL completely abolished the stimulatory effect on [3H]thymidine incorporation of epidermal growth factor (100 pg/ml) and PNA (25 micrograms/ml) and markedly inhibited the stimulatory effect of insulin (50 ng/ml). ABL (0.2 mg/ml) caused no cytotoxicity to HT29, MCF-7, and Rama-27 cells as measured by trypan blue exclusion, and inhibition of proliferation in HT29 cells caused by 50 micrograms/ml ABL was reversible after removal of the lectin. Binding studies with 125I-labeled ABL suggested a single class of binding site with an apparent Kd value of (4.12 +/- 0.29) x 10(-7) M with (3.6 +/- 0.3) x 10(7) binding sites/cell. A. bisporus lectin is a reversible noncytotoxic inhibitor of epithelial cell proliferation which deserves study as a potential agent for cancer therapy.

Retinoids are potent regulators of epithelial cell growth and differentiation. Recently, they have been demonstrated to be effective in the treatment of preneoplastic cervical lesions in which human papillomavirus (HPV) is expressed. To better understand the mechanism of the antineoplastic effect of retinoic acid on HPV-positive cells, the effects of retinoic acid on both normal and HPV-immortalized human ectocervical epithelial cell growth, epidermal growth factor (EGF) receptor level, and EGF receptor function were investigated. Both HPV-immortalized cells (ECE16-1) and normal ectocervical cells (ECE cells) are growth stimulated by EGF. ECE16-1 but not normal ectocervical epithelial cells are growth inhibited by trans-retinoic acid which attenuates the stimulatory effect of EGF on ECE16-1 cell growth. Retinoic acid reduces both EGF binding and EGF receptor protein levels in ECE16-1 cells but not in normal ectocervical cells. The reduction in EGF receptor binding and receptor protein levels in ECE16-1 cells is not associated with the induced secretion of a soluble EGF receptor ligand, altered EGF receptor affinity, receptor internalization, or decreased receptor stability. Interestingly, the level of EGF receptors is consistently elevated in the ECE16-1 cell line as compared to normal ectocervical epithelial cells. Investigation of a second HPV-immortalized cell line (ECE16-D1) and two other HPV-positive cervical carcinoma cell lines revealed similar elevated EGF-binding capacity and regulation by retinoic acid. In contrast, two HPV-negative cervical carcinoma cell lines demonstrated various EGF-binding levels but demonstrated no significant loss of EGF binding following retinoic acid treatment. Other normal cells and an SV40 large T-antigen-immortalized foreskin keratinocyte cell line, KER-1, had EGF receptor levels similar to the normal ectocervical epithelial cells, and no regulation by retinoic acid was observed. These data indicate that HPV immortalization may increase EGF receptor levels in ectocervical cells, elevating their sensitivity to growth stimulation by EGF, and that retinoic acid can possibly attenuate this increased responsiveness to EGF.

Vitamin A deficiency of respiratory tract epithelium results in the phenomenon of squamous cell metaplasia. The mechanisms by which vitamin A regulates airway epithelial cell growth and differentiation are not completely understood. In this study, we focused on the effects of vitamin A (retinol) on growth of human and non-human primate tracheobronchial epithelial (TBE) cells in culture. Retinol and its derivatives have little growth-stimulatory effect on TBE cells that are maintained in primary culture in a serum-free medium supplemented with 6 hormonal supplements: insulin, transferrin, epidermal growth factor (EGF), hydrocortisone, cholera toxin, and bovine hypothalamus extract. However, it was observed that retinol exhibited dose-dependent inhibition of TBE cell growth when EGF was removed from this serum-free culture condition. This inhibition can be reversed if EGF or the conditioned medium of primary TBE cells that are maintained in vitamin A-deficient condition is added. This type of EGF-retinol interacting phenomenon was not observed with the 5 remaining hormonal supplements. Analysis of 125I-labeled EGF binding shows a down-regulation of the high affinity binding sites (Kd = 0.09 nM) on TBE cells grown in the absence of vitamin A. These results suggest that TBE cells are capable of secreting an EGF-like growth factor in the absence of vitamin A. The possibility that transforming growth factor-alpha (TGF-alpha) is involved in this phenomenon is further examined by antibodies specific to TGF-alpha and its binding to an EGF-receptor. Using the TGF-alpha antibody, the presence of a TGF-alpha-specific antigen was found to be 3-fold higher in the conditioned medium obtained from the vitamin A-deficient cultures than that derived from retinol-treated cultures. Furthermore, the antibody neutralizing the TGF-alpha binding to an EGF receptor was able to reduce the DNA synthesis associated with the vitamin A deficiency. These results suggest that vitamin A plays an important regulatory role in the paracrine/autocrine secretion of EGF/TGF-alpha-like mitogen in TBE cell cultures.

Effects of simultaneous treatment with NaNO2 and butylated hydroxyanisole, catechol, or 3-methoxycatechol were examined in a rat multiorgan carcinogenesis model. Groups of 15 animals were given a single i.p. injection of 100 mg/kg of body weight diethylnitrosamine, 4 i.p. injections of 20 mg/kg of body weight N-methylnitrosourea, 4 s.c. injections of 40 mg/kg of body weight dimethylhydrazine, p.o. treatment with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in the drinking water for the first 2 weeks and p.o. treatment with 0.1% 2,2'-dihydroxy-di-n-propylnitrosamine in the drinking water for the next 2 weeks of the initial 4-week initiation period. Starting 3 days after the completion of these carcinogen treatments, animals were given diets containing 2% butylated hydroxyanisole, 0.8% catechol, 2% 3-methoxycatechol, or basal diet either alone or in combination with 0.3% sodium nitrite until week 28, when complete autopsy was performed. Histological examination showed that NaNO2 strongly enhanced development of forestomach lesions but inhibited that of glandular stomach lesions in rats simultaneously given catechol or 3-methoxycatechol with or without prior carcinogen exposure. 3-Methoxycatechol promoted esophageal carcinogenesis either with or without NaNO2, but promoting effects of catechol were evident only in the presence of NaNO2. In addition, treatment with NaNO2 after carcinogen exposure enhanced forestomach carcinogenesis. These results indicate that NaNO2 can modify phenolic antioxidant-induced cell proliferation and/or carcinogenesis, particularly in the upper digestive tract.

Despite an increasing incidence of human breast cancer, its etiology remains unknown. Since some environmental chemicals are stored in human breast fat and are rodent mammary carcinogens, determining the genotoxic potential of environmental agents in this key target tissue is important. An assay was developed for detecting genotoxic activity, as unscheduled DNA synthesis (UDS), induced by chemicals and UV radiation in early passage cultures of normal human mammary epithelial cells (HMEC) derived from 5 different women. In order to measure UDS in culture, reduction in the percentage of cells in S-phase was accomplished either by depriving the cells of epidermal growth factor and bovine pituitary extract or by contact inhibition of growth. Cultures were incubated with test chemicals for 24 h in the presence of [3H]-thymidine. UDS was quantitated autoradiographically as net grains per nucleus (nuclear grains minus cytoplasmic background, population average) with > or = 6 net nuclear grains considered in repair for any individual cell. A positive response was observed with UV radiation, benzo(a)-pyrene, aflatoxin B1, ethylmethanesulfonate, 1,6-dinitropyrene, 2-acetylaminofluorene, and tobacco smoke condensate but not 7,12-dimethylbenz(a)anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin. These results demonstrate that HMEC from all 5 women examined have the ability to metabolize a variety of environmental chemicals to DNA-reactive forms. Furthermore, some chemicals known either to cause mammary cancer in rodents or to be contaminants in human breast tissue are genotoxic in HMEC. A positive response in passage 9 cultures was observed only with direct acting agents, suggesting that HMEC may lose their metabolic capabilities in longer-term cultures. The HMEC UDS assay may be used to address the role of environmental agents in human breast cancer by determining whether chemicals are DNA reactive or metabolized to DNA reactive species in this critical target tissue.

Administration of the direct acting carcinogen N-methyl-N-nitrosourea (NMU) to 50-55-day-old virgin female rats on different days of the estrous cycle yields differential breast tumor biology (T. A. Ratko and C. W. Beattie, Cancer Res., 45: 3042-3047). One basis for these estrous cycle-dependent differences may be the duration of cell cycle stages of susceptible structures such as mammary terminal end buds or the quantity and duration of repair effected following adduct formation within these structures. The terminal end bud (TEB) epithelial cell cycle was characterized using pulse injections of [3H]thymidine (0.5 mCi/g body weight). On estrus, TEB epithelial cell cycle was significantly shorter (15.5 h) than on proestrus (19.9 h) and diestrus (18.8 h). The shorter duration in TEB cell cycle on estrus was likely due to a shorter TG1 (3-4 h) (P less than 0.05) since TS and TG2 did not differ between estrous cycle days. When NMU was injected 1 h after [3H]thymidine, the labeled mitotic wave within TEB of diestrus rats recovered approximately 2-3 h sooner than those given injections during proestrus (P less than 0.01), suggesting less initial damage or a slightly faster rate of DNA adduct repair. When [3H]thymidine was injected 1-5 days after the NMU, the percentage of labeled mitoses of rats given injections during diestrus and proestrus recovered to near normal 48 h after NMU, although the proportion of all cells labeled was still low compared to non-NMU-treated rats. The percentage of labeled mitoses and labeling of cells were normal 3 and 5 days after NMU. Rats receiving a carcinogenic but sublethal dose of NMU (5 mg/100 g body weight), followed by [3H]thymidine injection within 1 min, had one-half the intensity of thymidine incorporation into the terminal end bud DNA of non-NMU-treated rats. Unscheduled DNA synthesis was not demonstrable within the first 48 h following injection of NMU. The results support and extend the finding that rat mammary epithelial cell carcinogenicity of NMU is estrous cycle dependent and appears to be correlated with a differential response in the cell cycle of TEB (shorter at estrus) or delayed recovery in response to NMU (proestrus versus diestrus).

The carcinogenicity of certain nickel compounds is well known. We have previously shown that human kidney epithelial cells were immortalized by treatment with Ni(II) and in cooperation with the v-Ha-ras oncogene transformed the cells to acquire tumorigenicity in athymic nude mice. Immunocytochemistry and sequence analysis of DNA from the nickel-immortalized cells revealed abnormal p53 expression and a T----C transition mutation in codon 238. These data are consistent with the hypothesis that Ni(II)-induced mutation in the p53 gene can be involved in the escape from senescence of kidney epithelial cells.

The temporary maintenance of a rat liver epithelial cell population at confluence before passaging followed by periods of rapid proliferation resulted in the generation of spontaneous transformants after about 108 population doublings. The appearance of morphologically aberrant transformants correlated directly with an increased resistance of the population to the growth inhibitory effects of transforming growth factor beta 1 (TGF-beta 1). Clonal cell lines derived from the transformants were resistant to TGF-beta 1 dependent inhibition of DNA synthesis. These cell lines were also highly tumorigenic and aneuploid, with characteristic gross chromosomal abnormalities, and they expressed a number of phenotypic markers common to rat liver epithelial cells transformed by oncogenes or chemicals. In contrast, apparently normal looking cell lines cloned from the same population were nontumorigenic and near diploid, with few chromosomal abnormalities, and they were as sensitive to TGF-beta 1 as early passage normal rat liver epithelial cells. Morphologically normal late passage rat liver epithelial cells were sensitive to transformation by the DNA hypomethylating agent 5-aza-2-deoxycytidine, in contrast to earlier passage cells, and this transformation was accompanied by the development of resistance to the growth inhibitory effects of TGF-beta 1. These findings suggest that acquisition of resistance to the effects of growth inhibitors such as TGF-beta 1 is an important and possibly essential stage in the spontaneous transformation of rat liver epithelial cells.

Epidemiological and experimental studies suggest that dehydroepiandrosterone (DHEA), an adrenal cortical steroid, has chemoprotective properties. Rat colonic epithelium which had been induced to a premalignant state by the colonic carcinogen azoxymethane was used as a model for patients at high risk of colorectal carcinoma, and the efficacy of dietary DHEA for chemoprotection against tumorigenesis was evaluated. Ten-week-old male F344 rats (n = 100) were given 10 weekly s.c. injections of azoxymethane at a dose of 10 mg/kg/week. One day after the final dose of carcinogen, DHEA was added to the diet of 50 rats (0.5% DHEA chow), and the other rats were used as pair-fed controls. DHEA-fed rats lost body weight throughout the 17-week study, in contrast to their pair-fed controls. Serum DHEA in DHEA-fed rats at the end of the study was 6 times that of controls (120 +/- 30 versus 18 +/- 14 pmol/ml), and serum DHEA sulfate was 23 times that of controls (1311 +/- 13 versus 55 +/- 13 pmol/ml). Addition of DHEA to the diet produced no significant chemoprotection in our model. Tumor-related mortality was somewhat increased in DHEA-fed rats (20% versus 6% in week 16 of DHEA feeding, P not significant). The cumulative prevalence of left colonic tumors, identified by weekly colonoscopic examinations, was somewhat lower in DHEA-fed rats than in controls during weeks 10 through 13 (17% versus 33% in week 12, P not significant), but in week 14 the prevalence in DHEA-fed rats became similar to that in controls (39% versus 41%). Growth curves of autochthonous left colonic tumors, as assessed for 8 weeks by computerized image analysis of colonoscopic photographs, were similar for DHEA-fed and control rats. Prevalence, mean frequency, multiplicity, and diameter of colonic tumors at necropsy of colonoscopically negative rats in week 17 were somewhat lower in the DHEA-fed rats (e.g., prevalence of 47% versus 67%), but the differences from controls were not significant. Parameters of colonic epithelial proliferation after tritiated thymidine incorporation in DHEA-fed rats were similar to those in control rats (labeling index of 8.3 +/- 0.7% versus 8.4 +/- 0.6% in week 17), despite higher serum DHEA and DHEA sulfate levels. Our findings indicate that DHEA did not have significant postinduction chemoprotective activity against azoxymethane-induced colonic tumorigenesis in this model utilizing pair-fed controls. Further preclinical studies appear to be needed before dietary DHEA can be recommended for chemoprotection trials in patients with premalignant colorectal epithelium.

Induction of ornithine decarboxylase (ODC) enzyme activity occurs after exposure of hamster tracheal epithelial (HTE) cells to asbestos and the soluble tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Since active oxygen species are implicated as mediators of asbestos-induced biological responses studies here were designed to examine whether active oxygen species generated by asbestos or oxidants caused increased ODC activity. In confluent HTE cells, significant blockage of chrysotile or crocidolite asbestos-stimulated ODC activity occurred with simultaneous addition of catalase, but not superoxide dismutase, to medium. The addition of xanthine plus xanthine oxidase caused a dose-dependent increase in ODC activity, which was inhibited significantly after addition of catalase or mannitol, indicating that H2O2 was the principal oxidant produced in that reaction. Addition of phenazine methosulfate, a redox reagent used to generate superoxide, resulted in significant elevation of ODC, which was inhibited by addition of superoxide dismutase but not catalase. Hydrogen peroxide added to culture medium also caused a potent increase in ODC activity inhabitable by catalase. Hypochlorous acid caused increases in ODC activity, although the magnitude of this response was less than that observed with other oxidants. Therefore, although all active oxygen species examined triggered ODC, less reduced species (O2- and H2O2) were more proficient than OH or a halogenated oxidant. All oxidants, except HOCl, caused a significant increase in [3H] thymidine incorporation at 24 or 48 h after their addition to HTE cells. In comparative studies, exposure of HTE cells to either asbestos or xanthine plus xanthine oxide increased the level of ODC mRNAs proportionate to oxidant concentration and the extent of enzyme induction. Thus, data indicate that H2O2 plays a major role in asbestos-stimulated ODC induction and proliferation of epithelial cells of the respiratory tract by altering the regulation of a gene critical to proliferation.

The ability of three model carcinogens, 1,2-dimethylhydrazine, dimethylnitrosamine, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, to induce mutation in a novel in vivo assay in mouse intestine has been examined. The assay is based on mutations at the Dlb-1 locus which determines the tissue specific pattern of expressio of the binding site for the lectin Dolichos biflorus agglutinin. In C57BL/6J x SWR F1 mice Dlb-1 mutants are recognized as clones of epithelial cells not staining with a peroxidase conjugate of D. biflorus agglutinin. Chronic administration of 1,2-dimethylhydrazine (20 mg/kg/week s.c. for 10 weeks) induced Dlb-1 mutants, whereas administration of a single dose did not. Similarly, chronic dimethylnitrosamine treatment p.o. (0.001% in drinking water for 8 weeks) induced Dlb-1 mutants, but acute administration did not. In contrast, neither chronic nor acute treatment of the mice with 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline induced Dlb-1 mutations. The activities of 1,2-dimethylhydrazine, dimethylnitrosamine, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the Dlb-1 assay more accurately reflect their carcinogenic potential than do many in vitro bioassays.

In order to study the effects of perinatal exposure to estrogens on the developing reproductive tract, outbred female mice were treated neonatally (days 1 to 5) with varying doses of diethylstilbestrol (DES) and sacrificed from 1 to 18 months of age. Uterine adenocarcinoma was observed in a time- and dose-related manner after DES treatment; at 18 months, neoplastic lesions were seen in 90% of the mice exposed neonatally to 2 micrograms/pup of DES/day, while none was observed in the corresponding control mice. These DES-induced uterine tumors were estrogen dependent; when DES-treated mice were ovariectomized before puberty, no uterine tumors developed. As a marker for neoplasia, uterine tumors were transplanted and carried as serial transplants in nude mice. The transplanted tissue retained some differentiated uterine gland structure and function and also required estrogen supplementation for maintenance. Additional groups of neonatal mice were treated with various DES analogues (hexestrol and tetrafluorodiethylstilbestrol) and steroidal estrogens. The compounds were ranked according to developmental estrogenic potency (hexestrol greater than trifluorodiethylstilbestrol greater than DES greater than 17 beta-estradiol). The combined prevalence of uterine atypical hyperplasia and adenocarcinoma follows the order of estrogenic potency. The experimental induction of these tumors will provide the basis for additional studies in mechanisms of hormonal carcinogenesis.

The effects of transforming growth factor beta (type 1) (TGF-beta 1) on DNA synthesis, cell proliferation, and protein synthesis were examined in a series of v-raf-transformed rat liver epithelial (RLE) cells, which exhibit a range of transformed phenotypes. All of the transformed cells were relatively resistant to the growth-inhibitory effects of TGF-beta 1, compared to normal RLE cells and control cells infected with a helper virus. The more tumorigenic cell lines had very few surface receptors for TGF-beta 1 and showed no increase in the secretion of a number of specific proteins, including fibronectin, following TGF-beta 1 treatment. In contrast, the more normal-looking, less tumorigenic v-raf-transformed cells bound similar amounts of TGF-beta 1 as normal RLE and control cells and showed a similar pattern of TGF-beta 1-stimulated protein secretion. These findings suggest that the effects of TGF-beta 1 on cell proliferation and on the expression of certain secreted proteins are mediated through different mechanisms. Following transformation of RLE cells with v-raf, the signalling pathways controlling TGF-beta 1 growth inhibition are perturbed, while those involved in regulating the synthesis of certain proteins may remain intact. Thus, the escape from the various distinct biological effects of TGF-beta 1 may be an important stage in the progression of neoplastic transformation of RLE cells in vitro.

One of the major goals in cancer research and diagnosis is to identify in a tissue the population of actively dividing cells and their pattern of growth and to differentiate the proliferation patterns of normal and transformed tissues. We now describe a method for determining the proliferation pattern of any tissue (normal, diseased, or transformed), applicable in any mammalian species. This method is based on the fact that the transcription of histone H3 gene in mammalian cells is tightly coupled to DNA synthesis during cellular division. Resting cells or cells that just exited the cell cycle will have no detectable H3 mRNA. The presence of H3 mRNA in a cell is thus a good indicator of its proliferation status. We carried out in situ hybridization of H3 mRNA in hamster oral epithelia exhibiting a variety of altered growth patterns as a consequence of exposure to the chemical carcinogen, 7,12-dimethylbenz[a]anthracene to demonstrate the usefulness of this technique. This application does not require in vitro manipulation of tissues nor does it require the prior administration of a tracer. The proliferation pattern at a single moment in time instead of an accumulated pattern over a period of time is produced. Finally, since the technique of in situ hybridization can be applied to archival tissues, retrospective studies can be done. This application should find usefulness in a wide variety of experimental research settings, particularly cancer research.

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced arachidonic acid metabolism was investigated in dog urothelial cells. Primary cultures of dog urothelial cells were grown to confluency and evaluated in the presence or absence of overnight prelabeling with [3H]arachidonic acid. High-performance liquid chromatography analysis of media from TPA stimulated cells indicated that prostaglandin E2 (PGE2) was the major eicosanoid produced. Lipoxygenase products were not detected. Control cell media contained only arachidonic acid. Effects of selected inhibitors on TPA and exogenous arachidonic acid mediated increases in radioimmunoassayable PGE2 were assessed. Prostaglandin H synthase inhibitors (indomethacin and aspirin) prevented both TPA and arachidonic acid increases in PGE2. By contrast, inhibitors of phospholipases (quinacrine, W-7, and trifluoropromazine), protein synthesis (cycloheximide), and protein kinase C (staurosporine) prevented TPA but not arachidonic acid increases in PGE2. The latter agents also reduced TPA mediated increases in the release of total radioactivity from cells labeled with [3H]arachidonic acid. However, aspirin reduced the amount of 3H-prostaglandins formed with TPA. A calcium requirement was demonstrated when increases in radioimmunoassayable PGE2 elicited by TPA and the calcium ionophore A23187 were reduced with calcium depleted media. When epidermal growth factor in combination with either TPA or bradykinin was used, at least additive effects were observed with respect to release of [3H]arachidonic acid, 3H-prostaglandins, and radioimmunoassayable PGE2. These experiments suggest that separate pathways may be involved in enhanced arachidonic acid metabolism demonstrated with different agonists. For TPA, increased arachidonic acid release occurs by a calcium dependent process involving phospholipase(s), protein synthesis, and protein kinase C.

Aflatoxin B1 (AFB1) is thought to be an occupational risk factor for airway carcinogenesis where exposure to AFB1-laden grain dusts is common. Since activation of AFB1 is catalyzed by cytochromes P-450 associated with the smooth endoplasmic reticulum, we compared the response to AFB1 in cultured tracheal epithelium from species with abundant (rabbit and hamster) and scarce (rat and monkey) distributions of smooth endoplasmic reticulum in nonciliated tracheal epithelial cells. Explants from each species, incubated in medium containing 0.5 microM [14C]-AFB1 for selected intervals up to 24 h, were compared on the basis of binding of [14C]-AFB1 to tracheal DNA, amount and type of AFB1 metabolites in the medium, ultrastructurally determined population densities of epithelial cells, and distribution of bound material in epithelium as determined by autoradiographic grain densities. Cultures derived from rabbits were most active in metabolic conversion and formation of AFB1-DNA adducts, followed by those from hamsters, rats, and monkeys. Rabbit tracheal epithelium formed a significantly greater proportion of glutathione conjugates, while that from hamster formed a greater amount of AFB1-dihydrodiol, compared to rats and monkeys. The monkey formed significantly greater proportions of aflatoxin Q1 and the rabbit more aflatoxicol, compared to other species. There was selective degeneration and accumulation of labeled material in nonciliated cells in both rabbits and hamsters but not in rats or monkeys. Explants from rabbit tracheas were much more susceptible to cytotoxic injury and had higher autoradiographic grain densities than explants from hamsters. We conclude that the presence of smooth endoplasmic reticulum-containing nonciliated epithelial cells is qualitatively associated with the activation and toxicity of AFB1.

The serum factor inducing hemorrhagic necrosis of transplantable tumors [tumor necrosis factor (TNF)], and the macrophage hormone associated with cachexia in cancer and certain infectious diseases [cachectin] are known to be the same protein. Because an association may exist between TNF and the cachectic state, we wished to examine the effect of TNF on the permeability of epithelial barriers. We present data showing that TNF affects the tight junctional region between epithelial cells, lowering the transepithelial resistance and potential difference, and increasing the flow of solute between cells and across the epithelium. These effects are dose dependent, rapidly reversible, and inhibited by a monoclonal antibody to TNF-alpha. We suggest that the release of TNF at various sites throughout the body will cause a general breakdown in the barrier function of an epithelial cell sheet. This may relate to the cachexia observed in certain disease states. These findings are similar to our earlier published effects of phorbol esters and diacylglycerols on tight junctions, suggesting that protein kinase C activation may be involved.

We examined whether hyperproliferation of colonic crypt epithelium during cancer induction by N-methyl-N-nitro-N-nitrosoguanidine (MNNG), in rats on a low fat and calcium diet could be reduced by added calcium p.o. From the age of 4 weeks, 104 male Sprague-Dawley rats received a low fat (3.5%), low calcium (0.05% calcium ion), and low vitamin D (0.4 IU/g) diet. Sixty-four also had calcium salts, derived from either calcium lactate or solubilized calcium carbonate, added to their drinking water; therefore their total calcium intake was about 1% of daily diet. At age 12 weeks the rats were divided into 4 treatment groups: 8 rats, not receiving added calcium, had rectal saline instillations weekly (saline control group) and were sacrificed after a further 28 weeks; 3 groups of 32 rats each received intrarectal MNNG (1.5 mg) weekly. One group, not receiving added calcium, was the MNNG control group; while the second group also received added calcium lactate, and the third group received calcium carbonate. Groups of 24 were sacrificed periodically until 28 weeks of treatment. Rats were sacrificed and epithelial proliferation was estimated, 1 week after the last intrarectal instillation, by in vivo labeling with tritiated thymidine and measuring the ratio of labeled to total colonic crypt epithelial cells. The mean labeling index of the MNNG treated and added calcium groups were significantly higher (8.7-9.5%) than that of the saline controls (2.8%) only at week 28; however, it was then still significantly less than that of the MNNG controls not having added calcium (17.9%). Hyperproliferation, during induction of colonic cancer by MNNG in rats on a low calcium diet, can be reduced by a calcium enriched diet even in the presence of a low fat intake.

Heterotopic tracheal transplants of rats were exposed in vivo to 150 or 640 microng of 7, 12-dimethylbenz(a)anthracene (DMBA) delivered over 2 weeks, and the epithelium was studied in vitro in an attempt to identify changes in growth behavior during early phases of neoplastic development. Explants were made from the exposed tracheas, as well as from a series of controls, and the rate of epithelial outgrowth from the explants was measured. Secondly, the capacity of the outgrowths to survive as primary cultures and their ability to survive multiple in vitro passages were studied. During the initial planting, the rate of outgrowth was by far the greatest from the explants preexposed to 150 microng DMBA. Outgrowth from explants preexposed to 640 microng DMBA was sparse during the first planting but increased markedly during repeated planting when insulin- and hydrocortisone-supplemented medium was used. Establishment of outgrowth during repeated planting of carcinogen-exposed tracheal pieces was clearly hormone dependent. Control explants did not exhibit this hormone dependency. Primary cultures could be established from only three of six explants preexposed to 150 microng DMBA. These explants had been initiated in insulin- and hydrocortisone-supplemented medium. The primary cultures were successfully subcultured. Primary cultures were also established from five of five explants preexposed to 640 microng DMBA and cultured in hormone-supplemented medium. At least one cell line was obtained from each of the explants. To establish and maintain primary cultures from control tracheas required an enriched medium containing amino acid supplements, sodium pyruvate, and putrescine, as well as the hormone supplements. However, such cultures could not be subcultured. The primary cultures from the carcinogen-exposed explants and the subsequently developed cell lines all exhibited morphological characteristics of keratinizing squamous epithelium. These characteristics include: epithelioid cell morphology, multilayering and sloughing of orangeophilic squamous cells, and the presence of keratohyalin granules. These experiments demonstrate a markedly increased in vitro growth capacity of tracheal epithelium after a short in vivo exposure to carcinogen.

The effects of selenium on three mammary epithelial cell lines (YN-4, WAZ-2t, and CL-S1) grown in vitro were examined by immunocytochemical and transmission electron microscopy technique. The primary effect of selenium at the ultrastructural level was the appearance of electron-dense inclusions within the mitochondrial matrix. The mitochondrial inclusions were seen in all three cell lines, although most readily induced in YN-4 cells, the cell line which is most sensitive to selenium-mediated growth inhibition. Selenium at 5 x 10(-8) and 5 x 10(-6) M did not alter cytoplasmic microtubules or intermediate filament networks, as determined by immunocytochemical staining. Immunocytochemical staining for cytoplasmic filaments and microtubules, and transmission electron microscopy observations, supported the contention that cells from all three cell lines were epithelial in origin, since they contained abundant desmosomes and were uniformly positive for keratin intermediate filaments. Whereas line YN-4 was negative for vimentin intermediate filaments, a minority (5 to 24%) of the cells in lines CL-S1 and WAZ-2t stained positively. In addition, the tumorigenicity of these three cell lines was assessed by in vitro growth assays and in vivo transplantation assays. Cell lines YN-4 and WAZ-2t, but not line CL-S1, were tumorigenic in syngeneic mice. All tumors were mammary adenocarcinomas. Cytochalasin B-induced multinucleation assay and growth as multicellular spheroides correlated positively with in vivo tumorigenicity, whereas saturation density and growth in low Ca2+ medium were not correlated with tumorigenicity. It is speculated that one of the early effects of selenium-mediated growth inhibition may be a modulation of mitochondrial function.

Virgin female BALB/c mice were exposed in vivo to whole body gamma-radiation and/or to 7,12-dimethylbenz[a]anthracene (DMBA) p.o. Mammary epithelial cells were isolated and assayed for carcinogen altered cell populations both in vitro by an epithelial focus assay and in vivo by injection into cleared fat pads of syngeneic host mice. Five groups of mice were exposed as follows: (a) sham controls; (b) 50-rad gamma-radiation; (c) 100-rad gamma-radiation; (d) 75 micrograms DMBA; or (e) 50-rad gamma-radiation followed in 1 week by 75 micrograms DMBA. Mammary epithelial cells were isolated and assayed at 24 h and at 1, 4, 16, and 52 weeks after in vivo exposure. Four to 12 mice per treatment per time point were individually assayed. Altered in vitro growth potential was characterized by the proliferation of carcinogen exposed (but not control) cells as epithelial foci which persisted at least 12 weeks in primary culture. Epithelial foci which could then be subcultured at least four times were termed subculturable epithelial foci. Altered in vivo morphogenic potential was characterized by dysplastic or neoplastic growth in host fat pads. With increased time in situ between exposure and assay, cell populations emerged which exhibited both increased in vitro subculturability and enhanced tumorigenic potential including a host response upon injection in vivo. Further, combined radiation and DMBA resulted in higher frequencies of subculturable epithelial foci than either treatment alone. The relevance of these progressive cellular changes to the process of mammary tumor development is discussed.

Selective nutritive conditions were used to isolate normal epithelial cells from fibroblasts in primary cell cultures prepared from adult rat prostate. The pure population of normal epithelial cells proliferated at an exponential rate on a simple polystyrene substratum with doubling times of 35 to 50 hr for 10 to 12 days in the absence of high epithelial cell density, other cell types, or added extracellular matrix elements. Optimization of the nutritive environment allowed direct analysis of the hormone:growth factor requirements for sustained proliferation of the isolated epithelial cells in serum-free medium. An in situ videometric method was used to assay the effect of over 30 known hormones and growth factors on proliferation of the prostate epithelial cell population. The results revealed direct mitogenic effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, one or more unidentified factors from bovine pituitary, and possibly prolactin. No direct mitogenic effect of androgen on isolated prostate epithelial cells could be demonstrated. Radioimmunoassay of androgen in the primary cultures showed that endogenous androgen was about 34 pM on Day 1 of culture and thus probably too low to mask a response to exogenous androgen. Deletion of any single active growth factor did not reveal an androgen response. The results demonstrate a multihormonal control of normal prostate epithelial cell maintenance and proliferation without the direct participation of androgen.