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A human parotid gland adenocarcinoma cell line, with an intercalated duct cell phenotype of the salivary gland and expression of vasoactive intestinal polypeptide and amylase, was cultivated in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate (dB-cAMP). Morphological changes occurred; cells formed long cytoplasmic processes densely packed with ample microfibrils, as well as microtubules, and grew in a netlike appearance. In addition, it has been found by the immunofluorescence staining technique, immunoblotting, or immunoelectron microscopy that the cells treated with dB-cAMP express neurofilaments, neuron-specific enolase, synaptophysin, and HNK-1 antigen, as well as the alpha- and beta-chains of tubulin, whereas these antigens are not detected in untreated cells. The expression of vasoactive intestinal polypeptide detected diffusely in the cytoplasm of untreated cells was restricted to the cell membranes during the cultivation of cells in the presence of dB-cAMP, while expression of amylase persisted in the treated cells in a fashion similar to that in untreated cells. Moreover, both anchorage-independent and anchorage-dependent growth of the cells was markedly suppressed in the presence of dB-cAMP. After removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype and growth rate of the untreated cells. These findings indicate that reversible conversion into cells with phenotypic features of neuronal cells of a human parotid adenocarcinoma cell line occurs in growth medium containing dB-cAMP.

Normal epithelia and carcinomas of the human uterine cervix were studied by monoclonal antibodies chain specific for cytokeratins 4, 8, 10, 13, 14, 18, and 19. Most cells in 13 examined squamous carcinomas revealed a cytokeratin phenotype detected in ectocervical basal cells and endocervical subcolumnar reserve cells: 8+, 14+, 18+, 19+, 4-, 10-, 13-. We propose that these two cell types are closely related or identical and that squamous carcinoma of the cervix originates in this cell type. In more differentiated tumor cells cytokeratins 4, 10, and 13, which are present in suprabasal layers of the normal ectocervical epithelium, were coexpressed with basal cell cytokeratins. Thus, contrary to previous beliefs, all cytokeratins detected in carcinomas were also present in normal epithelium of uterine cervix. The cytokeratin profile of cervical adenocarcinomas corresponded to that of columnar endocervical cells (8+, 18+, 19+), although two of the three adenocarcinomas also expressed cytokeratin 4, which in the normal endocervix was detected in scattered single columnar cells only. The new monoclonal antibody DE-K14, specific for cytokeratin 14, proved a specific marker of subcolumnar reserve cells in the endocervix. It was also the only one that reacted with all cervical squamous carcinomas but with none of the cervical adenocarcinomas and, as such, has a potential value for pathological differential diagnosis of cervical tumors.

Cell surface receptors for laminin may play an important role in tumor migration and metastasis. To evaluate laminin receptor/laminin-binding protein expression in human colon carcinoma, surgical specimens of primary colon cancers and liver metastases were examined by blot hybridization of total RNA with a complementary DNA clone which encodes a Mr 32,000 human laminin-binding protein. The mRNA level of the laminin-binding protein was higher in primary colon carcinoma than in adjacent normal colonic epithelium in 20 of 21 cases. In all 6 cases of colon cancer liver metastases, the laminin-binding protein mRNA level was more than 3-fold greater in tumor than in adjacent normal liver tissue. The tumor/normal ratio of this laminin-binding protein mRNA expression in primary colon cancer has significant correlation with Dukes' classification (P less than 0.001). Our results suggest that mRNA expression of the laminin-binding protein may be a marker of human colorectal cancer progression and biological aggressiveness.

In view of advancements in treatment of certain hormone-dependent cancers with analogues of luteinizing hormone-releasing hormone (LH-RH), this study was undertaken to establish the presence and characteristics of receptors for [D-Trp6]LH-RH on the membranes of human endometrial cancer. Specific binding of [125I,D-Trp6]LH-RH was demonstrated in membrane preparations from 24 of 31 (77%) endometrial carcinomas and from 3 of 13 (23.1%) nonmalignant human endometrial specimens. Ligand binding was dependent on temperature, time, and plasma membrane concentration in a fashion expected of a peptide hormone. Mathematical analysis of the binding data showed that interaction of [125I,D-Trp6]LH-RH with the binding sites was consistent with the presence of a single class of high affinity, noncooperative receptors (Kd 9.88 +/- 4.59 x 10(-9) M; Bmax 0.70 +/- 0.14 x 10(-12) mol/mg membrane protein). The rates of association and dissociation were calculated to be 6.5 x 10(6) M-1 min-1 and 0.021 min-1, respectively. [125I,D-Trp6]LH-RH binding was not displaced by either unlabeled somatostatin or epidermal growth factor, but was displaced completely by native LH-RH. Using 125I-epidermal growth factor, specific, high-affinity receptors were also detected in membranes from 22 of 26 (85%) endometrial cancers and in all of 6 nonmalignant endometrial specimens (Kd 0.42 +/- 0.12 x 10(-9) M; Bmax 0.30 +/- 0.15 x 10(-12) mol/mg membrane protein). The potential functional role of the receptors for [D-Trp6]LH-RH in human endometrial carcinoma is not clear, but this finding provides a rationale for the use of therapeutic approaches based on LH-RH analogues in this malignancy.

Two types of human fibronectin have been detected by the reactivity of specific monoclonal antibody FDC-6: (a) those present in normal plasma and adult tissues, which do not react with FDC-6 (normal fibronectin or norFN); and (b) those present in fetal tissues and in tumoral cell lines, which do react with FDC-6 (oncofetal fibronectin or onfFN) (H. Matsuura and S. I. Hakomori, Proc. Natl. Acad. Sci. USA, 82: 6517-6521, 1985). We compared the distribution of norFN and onfFN in normal breast tissue (15 samples), breast fibroadenoma (ten samples), and breast adenocarcinoma (80 samples), using an immunofluorescence technique with monoclonal antibody FDC-6. A polyclonal antiserum against human normal fibronectin was used as a control. While norFN was diffusely present in normal gland and in benign and malignant tumors, onfFN was absent in normal gland and in benign tumors. In carcinomas, however, its presence was frequent, as we found it in 60% of cases. Among classical prognosis factors in breast carcinomas, onfFN distribution was significantly correlated with histological grade. Indeed, the presence of FDC-6 labeling was significantly linked with intermediary and high malignancy grades, while its absence was significantly linked with low malignancy grade. onfFN could be considered a marker of the neoplastic state; its immunohistological detection may represent a new prognosis factor in breast carcinomas.

SW-13 cells, derived from a human adenocarcinoma of the adrenal cortex, formed only a few small colonies when suspended in soft agar at low cell densities. The number and size of colonies increased dramatically following stimulation with serum-free medium conditioned by SW-13 cells, indicating the possibility of autostimulation in these malignant cells. Evidence is presented suggesting that SW-13 cells form progressively growing soft agar colonies upon stimulation by epithelial tissue-derived growth factor-like polypeptides. Both acid-ethanol extracts and conditioned media from three human carcinoma cell lines (A431, D562, and A549) caused similar increases in colony number and size of SW-13 cells. Extracts from 26 of 32 freshly excised human carcinomas and five freshly excised nonneoplastic human kidneys and one human lung stimulated soft agar growth of SW-13 cells as well. None of the nine extracts from nonepithelial human solid malignant tumors stimulated SW-13 cells. However, a benign nonepithelial tumor (uterine leiomyoma) caused a low level of soft agar growth of SW-13 cells. Cell extract from A204 human sarcoma cells and both conditioned medium and acid-ethanol cell extract from A375 human melanoma cells lacked SW-13 activity, whereas medium conditioned by A204 cells stimulated soft agar growth of SW-13 cells. Chemical and physical treatment data indicated that the epithelial tissue-derived growth factor-like substances are acid- and heat-stable polypeptides with disulfide bonds. The major peak of this activity had an apparent molecular weight of 20,000 to 22,000 and was clearly separable from transforming growth factors reported previously which stimulate colony formation by nontransformed mouse AKR-2B and rat NRK cells. The major peaks of SW-13, NRK, and AKR-2B activity could be separated by high-performance liquid chromatography. This SW-13 activity induced irreversible anchorage-independent growth of SW-13 cells and an increase in DNA synthesis as measured by [3H]thymidine incorporation.

The structure of colonic mucin, which is thought to be important in several diseases, including ulcerative colitis and colon cancer, is poorly understood. Mucin was isolated from nude mouse xenografts of the LS174T colonic adenocarcinoma cell line by gel filtration and CsCl density gradient centrifugation. The isolated mucin had a high content of threonine, serine, and proline, with 28% of the total amino acids O-glycosylated. The carbohydrates present were fucose, sialic acid, galactose, N-acetyl-glucosamine, and N-acetyl-galactosamine in the ratio of 0.4:1.5:1.0:0.9:1.4. Rabbit antibodies were prepared that recognized primarily protein-dependent determinants. By DEAE-cellulose chromatography, the purified mucin was found to be heterogeneous, with three major components that had small differences in carbohydrate composition. LS174T was antigenically and chromatographically similar to mucins in colon cancer tissue specimens and in nonmalignant colonic mucosae.

The association of N1-acetylspermidine with human colorectal adenocarcinomas has been evaluated in this study. Free polyamines and their monoacetylated forms in adenocarcinomas, adenomas, and apparently healthy mucosae were determined using high-performance ion-exchange chromatography. The N1-acetylspermidine levels in well- and moderately differentiated adenocarcinomas were 27.30 +/- 3.13 (S.E.) (n = 99) and 22.86 +/- 3.60 (n = 22) nmol/g, wet weight, respectively. These values were significantly higher than those of benign adenomas (5.38 +/- 0.85 nmol/g, n = 31) and of control mucosae. The N1-acetylspermidine levels in control mucosae on the oral and anal side of adenocarcinomas were 5.84 +/- 1.44 (n = 57) and 7.92 +/- 2.89 (n = 50) nmol/g, respectively; no significant difference was observed between control mucosae and adenomas. The mean levels of three polyamines, putrescine, spermidine, and spermine in both adenomas and adenocarcinomas were about twice as high as those of control mucosae. The molar ratios of spermidine to spermine were significantly greater in both adenomas and adenocarcinomas than in control tissues. There was no obvious correlation between the free polyamine concentrations and the degree of malignancy of the colorectal tumors. These results suggest that the metabolism of N1-acetylspermidine in colorectal adenocarcinomas is quite different from that in adenomas and in nonneoplastic mucosae and that N1-acetylspermidine can be a promising biochemical marker of cancer in the human large intestine.

The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-[2-3H]mannose or L-[5,6-3H]fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with [2-3H]mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with [2-3H]mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-[1,6-3H]glucosamine and L-[1-14C]fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced 3H-labeled N-acetylglucosamine and N-acetylgalactosamine. This high molecular weight glycopeptide fraction was susceptible to mild alkaline borohydride reduction, yielding a mixture of labeled oligosaccharides which contained N-acetylgalactosaminitol. Thus, the HCT-8R cells are expressing fucosylated mucin-type glycoproteins on their surface.

Electron spin resonance (ESR) studies at -130 degrees have been made on frozen samples of normal human cervix and uterus and on frozen samples of various pathological conditions of the cervix and uterus including fibroleiomyoma and carcinoma. Fifty-five samples of normal cervix and endometrium, 40 samples of nonmalignant disturbances, 15 benign tumor samples, and 20 malignant samples were studied. Very strong ESR signals were seen in frozen powders and frozen intact samples of normal cervix and endometrium and in nonmalignant gynecological conditions. In many cases, the ESR signal was greatly decreased or even undetectable in cancer samples. The substance(s) responsible for the ESR signal in frozen intact tissue (g = 2.11 to 2.15) is decreased in concentration when the sample is ground to powder under liquid nitrogen, and an anisotropic signal (g = 2.002 to 2.035) then becomes much more evident. The ESR signals in intact and in powder samples are sensitive to temperature variations; the signals disappear around 0 degrees, and only the intact samples show significant recovery of signal on recooling. The anisotropic g values and temperature sensitivity in the powders may result from an organic peroxy radical that is more strongly associated with a metal ion in intact samples.

The keratins and other cytoskeletal proteins expressed by normal, preneoplastic, and malignant mammary tissues in BALB/c mice and by cells in primary cultures established from these tissues were analyzed and compared. The preneoplastic lesions were hyperplastic alveolar nodules (HAN) derived originally from mice treated by hormonal stimulation (D2), exposed to a chemical carcinogen (C4), or spontaneously expressing mouse mammary tumor virus (CV2) and maintained by serial transplantation. All tumors were mammary adenocarcinomas which developed as primary neoplasms from the HAN outgrowth lines. Cytoskeletal extracts were prepared from the tissues and cultured cells and subjected to two-dimensional polyacrylamide gel electrophoresis. Comparison of the major polypeptides in the normal and abnormal tissue extracts revealed considerable similarities in the cytoskeletal profiles. Three basic and seven acidic polypeptides ranging in molecular weight from 40,000 to 90,000 were regularly identified. However, notable differences were also found. A Mr 55,000 keratin (IEF 55) was prominent in one HAN, the D2, and all tumor tissues but not in normal gland. Likewise, a Mr 46,000 polypeptide (IEF 46), which has been tentatively identified previously as a keratin, was absent in normal epithelium but present in all abnormal tissues except the C4 and CV2 HAN. A Mr 58,000 polypeptide (NEPHGE 58) was not detected in normal gland or the C4 lesions but was found in all other abnormal tissues. The overall pattern of polypeptides in cytoskeletal extracts from normal and abnormal mammary cells in primary culture resembled that of the corresponding tissue but also had important differences. In all cell cultures, IEF 46 and IEF 55 were major species, while the larger and more basic components were markedly reduced. A Mr 56,000 polypeptide (NEPHGE 56) was detected only in C4 HAN and C4 and CV2 tumor cells. Trace or small, variable amounts of a Mr 57,000 basic keratin (NEPHGE 57) were present in normal and D2 tissues and cultured cells. NEPHGE 57 was dramatically increased in C4 and CV2 tissues and cultured cells and may be related to expression of squamous metaplasia and keratinization which are characteristic of these lesions. Although production of IEF 46 and IEF 55 may be associated with neoplastic progression of mammary epithelium, particularly in vivo, the association is not exclusive since normal cells express these polypeptides when grown in primary culture. In addition, correlations between altered keratin expression and the mode of induction of the mammary lesions were not obvious.

The purpose of this study was to compare the expression of O-acetylated sialic acids on normal colonic epithelial cells to that on primary and metastatic human adenocarcinoma of the colon and rectum. In 24 cases, the relative percentages of biosynthetically labeled non-, mono-, di-, and tri-O-acetylated sialic acids were measured after hydrolytic release, separation, and identification by paper chromatography. In one case, the presence of di- and tri-O-acetylated sialic acids was confirmed by fast atom bombardment-mass spectral analysis. Differences were observed in the expression of sialic acids between normal colonic epithelium, "uninvolved" colon mucosa remote to a colonic adenocarcinoma, and colonic adenocarcinoma. The levels of mono- and tri-O-acetylated sialic acids accounted for the difference in the ratios of sialic acids expressed between normal and "uninvolved" colonic mucosa, while the total amount of O-acetylation was unchanged. However, no difference was observed in the relative amounts of non- and O-acetylated sialic acids between either fresh and tissue culture-established colon carcinomas, or fresh and tissue culture-established liver metastasis derived from carcinoma of the colon. The relative expression of these O-acetylated sialic acids molecules appears to vary according to tissue type. This study suggests that individuals with adenocarcinoma of the colon express a field defect resulting in abnormal ratios of O-acetylated sialic acids.

Gastrin-releasing peptide (GRP) is now known to be a very common product of small cell lung carcinoma (SCLC). With the aim of investigating the possible role of this peptide as a tumor marker of SCLC, we have developed a sensitive radioimmunoassay system for plasma immunoreactive GRP using immune-affinity chromatography for plasma extraction. Plasma immunoreactive GRP levels in control subjects were determined by using 15 ml of plasma as the starting material (minimum concentration detectable, 0.8 pg/ml). The levels in 10 control subjects were (mean +/- SD) 1.2 +/- 0.27 pg/ml; range, 0.86-1.7 pg/ml. This assay system was applied for the clinical use by using 3 ml of plasma as the starting material (minimum concentration detectable, 4.0 pg/ml). Plasma immunoreactive GRP levels were elevated in SCLC patients at frequencies of 71% in patients with limited disease and 80% in those with extensive disease. Furthermore, a change in the level showed excellent correlation with the therapeutic response. In six patients with complete response who had had elevated levels before treatment, the levels decreased to an undetectable range when the tumor disappeared, and they remained undetectable until 1 month later, when the patients were judged to have achieved complete response. In the partial response group, plasma immunoreactive GRP levels had decreased to an undetectable level in two of three patients, when the patients achieved partial response. In four patients with progressive disease, plasma immunoreactive GRP levels were elevated at the time of the progressive disease judgment, when compared with levels before treatment. The levels in 21 patients with non-SCLC (10 with adenocarcinoma, seven with squamous cell carcinoma and four with large cell carcinoma) were not elevated. These results indicate the plasma immunoreactive GRP level as a useful tumor marker in SCLC patients. It is now believed that GRP can function as an autocrine growth factor for SCLC. The present study suggests that the possible autocrine growth factor could serve as a reliable tumor marker for cancer patients.

Modal DNA (ploidy) and sensitivity of DNA in situ to denaturation by acid have been analyzed by flow cytometry of 10 colorectal adenomas and 35 adenocarcinomas; 39 normal mucosa samples served as controls. A new method was developed to denature DNA in chromatin of the freshly isolated, intact, and unfixed individual cell nuclei from surgically resected material. The sensitivity of DNA denaturation (T alpha) was assayed by metachromatic staining with acridine orange and calculated as a ratio of the alpha t index of the tumor sample to the alpha t index of normal mucosa; the alpha t index is that fraction of DNA, following treatment at pH 1.4, that stains metachromatically with acridine orange at pH 2.6. All adenomas were diploid and in nine of 10 the T alpha value was close to 1.00, indicating no difference from control specimens in DNA sensitivity to denaturation. Forty-nine% of adenocarcinomas were aneuploid. Forty-six% of adenocarcinomas differed from normal in sensitivity of DNA to denaturation; the T alpha value was lower than 0.90 indicating that chromatin of the tumor cells was more resistant to denaturation than control cells. There was no correlation between sensitivity to denaturation of DNA and incidence of aneuploidy. However, there was a correlation between T alpha and the pathologically determined stage of disease. There was increased resistance to denaturation in 58% of tumors classified as Dukes' C/D stage, in 36% of tumors classified as Dukes' B, and in 20% classified as Dukes' A stage of the disease. Statistical analysis of these results revealed significant differences between distributions of T alpha in noninvasive (Adenomas and Dukes' A) versus invasive (Dukes' B and C/D) tumors with level of significance at P = 0.02. The data suggest that acid denaturation of DNA in situ may be a valuable adjunct in assessing the biology of colon cancer. The molecular basis for this phenomenon is discussed.

We have investigated the biosynthesis and carbohydrate structure of fibronectin secreted by two rat colon carcinoma cell lines. The cell line Prob yields progressive tumors after s.c. injection in syngeneic BD IX rats while tumors developed by Regb cells disappear after 20 days. No difference was observed in the fibronectin biosynthesis from both cell lines; however, the glycosylation degree was higher in Regb than in Prob cells indicating probable differences in the posttransductional process. The analysis of the glycosylation nature shows that fibronectin doesn't bear O-linked carbohydrate chains. The fibronectin of progressive Prob cells is more sialylated than that of the regressive Regb ones. In addition, the tri- and tetraantennary glycans are more important in Prob, while the fucosylated triantennary glycans are three times higher in Regb cells. These differences in the glycosylation state of the fibronectins could explain their differential susceptibility to the proteases treatment. In fact, the low glycosylated fibronectin from the progressive Prob cells was more rapidly degraded by several proteases than that of regressive Regb cells. The identification of the specific sites of proteolytic cleavage by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the cell attachment domain as well as the collagen binding domain of Regb cell fibronectin are particularly protected against proteolytic degradation.

Immunohistochemical localization of estrogen receptor (ER) using specific monoclonal anti-human estrogen receptor antibody, H222, with an immunoperoxidase technique was performed on fresh frozen tissue derived from 100 endometrial adenocarcinomas. Immunohistochemical evaluation incorporated both intensity and distribution of staining. In all cases, H222 localized in the nucleus of target cells. A significant quantitative relationship was shown between histological score (H-Score) and the biochemical analysis of ER content in tissue homogenates (r = 0.65, P = 0.00001). Excellent sensitivity (92%) and specificity (93%) were observed for the comparison of H-Score to the biochemical assay. Significant ER localization was present in stromal and myometrial elements, component H-Score of which correlated weakly with component H-Scores of malignant epithelial elements. Divergent receptor localization in stromal and myometrial versus malignant epithelial elements suggests that biochemical assays of endometrial carcinoma specimens may not reflect cancer-relevant receptor content. The data presented here suggest that the immunoassay of ER using H222 monoclonal antibody provides additional histochemical information to complement conventional analyses of endometrial adenocarcinomas.

Digestion of primary breast cancers and their metastases with collagenase yields cell clusters which can be selectively isolated from stromal cells and from the less malignant-looking epithelium of the primary tumors by their failure to attach as rapidly to collagen gel. Continued passage in culture of one preparation of cell clusters has yielded a continuously growing cell strain, termed Ca2-83. This strain continues to grow mainly as cell clusters with doubling times of 10 to 14 days, although some clusters eventually adhere to plastic substrata. Two morphological extremes of cell were observed, smaller polygonal or cuboidal cells and larger, often-multinucleated cells which contain fat droplets. Cell clusters grew in a gland-like pattern similar to those of the original carcinoma and formed small nodules in 50% of recipient nu/nu mice. Both morphological forms of Ca2-83 in culture or in tumor nodules stained immunocytochemically with epithelial cell-specific antisera to epithelial membrane antigens and to human keratins but not to laminin or actin. Cultures of Ca2-83 failed to synthesize laminin under conditions where its synthesis was observed in a rat myoepithelial cell line. Ultrastructural analysis of the cell clusters has identified microvilli coated with epithelial membrane antigens and junctional complexes typical of secretory epithelia in both morphological forms, but no characteristics of myoepithelial cells or basement membranes were observed. The DNA content of the cultures increased in response to serum, a bovine pituitary fraction, and insulin. Numbers of cell clusters were also increased in the presence of culture medium exposed to preadipocytes, myoepithelial- or mesothelial-like cells/stromal cells, or to prostaglandin E2.

Total neutral (nonacidic) glycolipid fractions have been isolated from a gastric adenocarcinoma and the surrounding normal gastric tissue removed at laparotomy of a Blood Group B human individual. Each glycolipid mixture was fractionated by silicic acid column chromatography into ten different partly purified glycolipid fractions. These fractions were analyzed by thin-layer chromatography and tested for Blood Group A and B activity. Blood group B activity was found in several fractions from both tumor and normal tissue. Two of the tumor glycolipid fractions reacted with some batches of commercial anti-A antisera, but other antisera tested did not react. No such Blood Group A reactivity was found in the fractions from the normal gastric tissue. The two Blood Group A-active tumor glycolipid fractions were methylated and methylated-reduced, and these two derivatives were analyzed by mass spectrometry. It was shown that these two fractions were mixtures of glycosphingolipids with five to nine sugars. The dominating glycosphingolipids were blood group Leb and B-like hexaglycosylceramides, a B-similar heptaglycosylceramide with an additional fucose, an H-like heptaglycosylceramide, and a Leb-like octaglycosylceramide. Evidence for small amounts of a Blood Group A-similar heptaglycosylceramide with an additional fucose was also found. The finding of a Blood Group A-similar glycolipid in a fraction which reacts with some anti-A antisera is the first chemical evidence for a heterolog blood group antigen in human cancer which has previously been found by histoimmunological techniques. The clinical significance of this finding is discussed in relation to diagnostic procedures and immunotherapy.

The adenocarcinoma cell line HSG from human salivary gland, which proliferates in vitro or in nude mice, was examined by the immunoperoxidase method for the expression of three different types of intermediate-sized filaments (IFs) and of specific antigens such as carcinoembryonic antigen, S-100 protein, secretory component, lactoferrin, myosin, tropomyosin, and actin. The cultured HSG cells were found to express three different types of IFs defined by antibodies to keratin, vimentin, and desmin. In HSG cells proliferating in vitro at 34 degrees C and 37 degrees C but not at 39 degrees C, the expression of tropomyosin and carcinoembryonic antigen was observed, although myosin and S-100 protein were not detected. The expressions of actin, lactoferrin, and secretory component were restricted to cultured HSG cells at 39 degrees C and 37 degrees C, respectively. Transplantation of HSG cells into nude mice resulted in the establishment of a nude mouse system with malignant characteristics such as invasion and metastasis. The expression of IFs in the primary tumors was restricted to keratin and desmin IFs, whereas coexpression of keratin, vimentin, and desmin IFs was observed in some neoplastic cells present in the metastatic tumors in regional lymph nodes and lung. In addition, expression of actin, myosin, tropomyosin, and S-100 protein was found in the metastatic tumors, whereas myosin and S-100 protein were not detected in the primary tumors. Moreover, the metastatic tumors were almost occupied by the neoplastic cells with oncocytic changes, although oncocytic change was not found in the cultured HSG cells and their primary tumors.

A study was undertaken to observe morphological changes and viral morphogenesis during the growth of the mouse preputial gland tumor ESR 586. The acinar cells of the normal preputial gland have an extensive agranular endoplasmic reticulum and Golgi apparatus and large lipid droplets. No viral particles are present. During tumor growth, and numerous lipid droplets never attain the size of those found in the normal gland. There is a decrease in the Golgi apparatus and agranular endoplasmic reticulum and an increase in the rough endoplasmic reticulum which could reflect a change in composition of tumor secretory product from the normal gland. Indeed, there is a decrease in triglycerides as the tumor ages and an increase in the sterol esters and waxes. In addition, intracisternal A-particles are observed budding from thickened endoplasmic reticulum membranes. Thickening of these membranes occur early in A-particle formation. One side of the membrane is first thickened while the opposing membrane appears is first thickened while the opposing membrane appears morphologically unaffected. The thickening of the affected membrane is initially confined to the outer (cytoplasmic) face of the membrane. In the older tumor, both opposing membranes of the reticulum are thickened and can assume an elongated whorled pattern.

Using c-Ha-, c-Ki-, and c-N-ras-specific probes in a RNA-RNA hybridization assay we found enhanced expression of c-Ha-ras protooncogene in stomach adenocarcinomas relative to nonneoplastic epithelium, whereas little or no transcription of either c-Ki- or c-N-ras was detected. Enhanced levels of c-Ha-ras RNA expression were detected in all of the adenocarcinomas examined. Hybridization with c-Ha-ras was also detected in nonneoplastic gastric epithelium adjacent to carcinoma, although the labeling was less intense than that of carcinoma cells. More extensive analysis of the c-Ha-ras p21 expression was then carried out in formalin-fixed, paraffin-embedded tissue sections and extracts from surgically resected stomach tissues using monoclonal antibodies (MAbs) RAP-5 and Y13-259. The data obtained from the immunohistochemical studies were consistent with the results of in situ hybridization assay. Adenocarcinomas were much more reactive with MAb RAP-5 than benign and normal tissues, and the majority of carcinomas demonstrated increased expression of c-Ha-ras p21. Quantitative liquid competition radioimmunoassays using MAb Y13-259 also demonstrated significantly higher levels of c-Ha-ras p21 in extracts from stomach adenocarcinomas than those from normal mucosae. No strict correlation was found between ras p21 expression and the degree of tumor differentiation or histological type. Although advanced carcinomas generally demonstrated higher levels of ras p21 than early carcinomas, no correlation among advanced carcinomas and ras p21 levels was observed in relation to depth of tumor invasion to the muscularis propria, subserosa, or serosa. Benign lesions, in comparison, were much less reactive with MAb RAP-5 than carcinomas. Among the benign lesions tested, dysplastic lesions were more reactive than nondysplastic lesions. Normal stomach mucosa was generally nonreactive with the exception of parietal cells. Our results indicate that transformation of the stomach mucosa from benign to malignant phenotype is associated with an increase in c-Ha-ras p21 expression.

In studies aimed at developing monoclonal antibodies against lung adenocarcinomas, we produced a murine monoclonal antibody designated 130-22 by immunizing mice with lung cancer cells. Since in immunoperoxidase staining experiments this antibody was reactive not only with lung adenocarcinomas but also with ovarian carcinomas, we examined its relationship to the ovarian cancer marker CA125, an antigen recognized by monoclonal antibody OC125 produced by immunization of mice with ovarian carcinoma cells. Although CA125 antigen was adsorbed by 130-22 antibody, 125I-labeled 130-22 did not compete with OC125, indicating that although these two antibodies recognized CA125 antigen, they reacted with separate antigenic determinants. The antigen defined by both antibodies was thought to be heat-labile glycoprotein with a molecular weight of over 1,000,000. A series of immunoradiometric assays was developed using combinations of two monoclonal antibodies in a simultaneous forward sandwich mode. Mixed monoclonal antibodies may provide a more sensitive assay for the detection of CA125 than the homologous assay, in which OC125 was used both as a tracer and as a catcher. These results indicate that CA125 is an antigen with two separate epitopes present in both ovarian and lung adenocarcinomas and that combination use of monoclonal antibodies reactive with different antigenic determinants will give certain advantages to the immunoradiometric assay of cancer markers.

The aim of this study was to investigate the effects of estradiol and tamoxifen (TAM) on the growth of human endometrial carcinomas in athymic mice. Tissues from primary tumors were implanted into estradiol-treated mice. In passage 2, animals were treated with (a) placebo, (b) estradiol, (c) estradiol plus TAM, and (d) TAM alone. The size of the tumors was measured weekly. Estrogen receptors (ER) were determined with the dextran-coated charcoal method and/or ER enzyme-linked immunoassay. Progesterone receptors were measured with the dextran-coated charcoal technique. Of 16 primary tumors, 2 grew in the athymic mice and were studied further. Tumor EL was positive for ER (145 fmol/mg protein) and progesterone receptors (993 fmol/mg protein). Tumor EL in passage 2 was not significantly stimulated by estradiol, but was stimulated by a combination of estradiol and TAM. Treatments (estradiol, estradiol plus TAM, or TAM) all increased tumor growth in passage 3. Tumor BR and a metastasis BR-MET were ER and progesterone receptor negative, applying dextran-coated charcoal, ER enzyme-linked immunoassay, and immunocytochemistry. The BR and BR-MET cells contain the complete ER gene but do not express any measurable amounts of ER mRNA as quantitated by Northern blot analysis, using a complete ER complementary DNA probe. In all animal passages the growth rate was significantly higher in estradiol-treated mice compared with the control. TAM alone had some growth stimulatory effect, but much smaller than observed in the estradiol group. TAM inhibited estradiol-stimulated growth. These results suggest that estradiol and possibly TAM are capable of stimulating tumor growth in the athymic mice independently from ER, potentially through a host-mediated mechanism.

The transplantable hormone-responsive rat mammary adenocarcinoma 13762NF was dissociated with collagenase and hyaluronidase. Cells were cloned directly or lines were established from mass cultures and cells from these lines were cloned. Clones differed in cellular morphology, colony morphology on plastic or in collagen gel, growth rate, growth response to hormones, and hormone receptor levels. Growth response to prolactin, estradiol, progesterone, cortisol, and epidermal growth factor (EGF) was determined by culturing the cells within collagen gel and using a serum-free medium base of DME/F12 (1:1) with insulin, linoleic acid, and BSA. The clones varied in their hormone responses, with all 20 of the clones tested responding to cortisol in combination with EGF. Some clones would respond to EGF, cortisol, or progesterone when used alone. None of the clones tested could be stimulated by prolactin or estradiol. Receptor levels for estradiol, progesterone, glucocorticoids, and EGF were assessed in 3 selected clones differing in their hormone responsiveness. Receptor levels appeared to correlate with hormonal sensitivity. Selected clones transplanted into female F344 rats produced carcinomas with histopathologies similar to the original tumor.

Estradiol and progesterone receptors were assayed in tumors from 79 patients with primary colorectal and 56 patients with stomach adenocarcinomas. Eighteen of 79 colorectal cancers contained estradiol receptor, while 34 specimens were positive for progesterone receptor. In stomach cancer, the positive samples were 8 for estradiol and 14 for progesterone receptors. In both types of tumors, the Kd was in the range of 10(-10) M for estradiol and 10(-9) M for progesterone receptor, respectively. In colorectal adenocarcinomas, the presence of progesterone receptor seems to be partially correlated to the presence of estradiol receptor while, in stomach tumors, this correlation is lost. The positivity of at least one receptor in colorectal cancers is higher in the female sex. The contrary occurs for stomach cancer. Sucrose gradient centrifugation showed that cytoplasmic estradiol receptor of stomach cancer sedimented at 8S or 4 to 5S at low ionic strength. The isoelectric point of stomach cancer estradiol receptor is 6.5.

In vitro, neopterin, a pyrazinopyrimidine compound, is excreted by human monocytes-macrophages after induction by supernatants from activated T-lymphocytes or by recombinant gamma-interferon. In vivo, it represents a noninvasive test for activation of cellular immune reactions. To evaluate the prognostic value of pretherapeutic urinary neopterin levels and of serial neopterin measurements during follow-up in women with cervical cancer, 1088 urine specimens from 186 consecutive patients were analyzed. Clinical assessments were made without knowledge of the results of neopterin assays (a "blinded" assessment). During the observation period (June 1980 to March 1984), 27 relapses, 18 metastases, and 26 deaths were seen. The prognostic significance of pretherapeutic neopterin and other possible prognostic clinical and laboratory parameters was tested by the univariate and multivariate Cox proportional hazards model using a stratification according to stage and surgical treatment. The combination of age at diagnosis, pretherapeutical hemoglobin, leukocyte count, and neopterin was found to predict survival best. On the basis of this result, risk groups were identified exhibiting markedly different survival behavior. A highly significant association was found between serial neopterin measurements and the risk for a relapse, metastasis, or death. The data suggest that urinary neopterin levels might be a useful adjuvant parameter in monitoring women with cervical cancer.

The levels of two prostaglandins (prostaglandins E and F) have been determined in a series of murine mammary lesions ranging from preneoplastic, hyperplastic alveolar nodules to highly metastatic adenocarcinomas. A highly positive correlation was seen between high levels of prostaglandin E and high tumorigenicity and metastatic potential. In addition, spontaneous metastasis of two highly metastatic tumors was partially inhibited by p.o. administration of indomethacin from the time of s.c. tumor transplantation until removal of the primary tumor at a limited size. Further, mammary tumor cells of differing metastatic potential were susceptible to polyinosinic-polycytidylic acid activated spleen lymphocytes in vitro. Cells of metastatic tumor lines (410.4 and 66) were more resistant to killing than were cells of two non-metastatic tumor lines (168 and 410). The sensitivity of all target cells was increased when endogenous prostaglandin synthesis was prevented by the addition of indomethacin (1 microM) but was not affected by the lipoxygenase inhibitor nordihydroguaiaretic acid.

The expression of cytokeratins (CKs) was investigated in cell lines and clones established from the rat 13762NF mammary adenocarcinoma tumor and its spontaneous lymph node and lung metastases. Two-dimensional polyacrylamide gel electrophoresis of intermediate filament-enriched protein fractions from cultured cells revealed that clones established from spontaneous metastases contained three CKs (Mr approximately 54,000, approximately 52,000, and approximately 40,000) characteristic of simple epithelia and two CKs (Mr approximately 51,000 and approximately 47,000) characteristic of stratified epithelia. CK expression varied qualitatively and quantitatively between the different metastasis-derived cell clones. In contrast, cell clones established from the original mammary fat pad tumor expressed low or undetectable levels of CKs. Western blot analyses with a panel of anti-CK antibodies with defined specificities confirmed the observations. One-dimensional polyacrylamide gel electrophoresis of whole-cell lysates and intermediate filament-enriched extracts were transferred and probed with the panel of antibodies. The relative expression of individual CKs varied according to the cell line or clone examined and environmental conditions (low versus high passage and in vitro versus in vivo growth), whereas the amount of total CKs expressed relative to total cell protein varied according to cell line or clone and growth conditions.

The presence of an estrogen-regulated protein with 24,000 molecular weight has been studied in 47 patients with endometrial carcinomas and in 29 patients with cervical carcinomas in order to correlate its presence with that of estrogen receptors (ERs) and progesterone receptors (PgRs). In the cytosol tumor samples the Mr 24,000 protein was detected by the Western blot technique using a monoclonal antibody (C11), while the presence of ER and PgR was studied by the one-point dextran-coated charcoal assay. In the tumor tissue sections immunohistochemistry was applied to detect Mr 24,000 protein, ER, and PgR; in these cases monoclonal antireceptor antibodies (H222 and mPRI) were used to localize the receptor proteins. In endometrial and endocervical adenocarcinomas the presence of Mr 24,000 protein correlated significantly with that of ER (P less than or equal to 0.05) in the cytosol samples; when the evaluation was performed in the tumor sections, the presence of Mr 24,000 protein correlated with that of ER (P less than or equal to 0.005) and PgR (P less than or equal to 0.05) as well. The study also showed that almost 70% of the well-differentiated adenocarcinomas had ER, PgR, and Mr 24,000 protein. In 25% of the endometrial adenocarcinomas examined the tumors were associated with normal, proliferative, and hyperplastic endometrium; in these cases the presence of ER, PgR, and Mr 24,000 protein was evaluated by immunohistochemistry in the malignant and nonmalignant endometrium. On the other hand, there was a lack of correlation between Mr 24,000 protein, ER, and PgR in the squamous carcinomas of the uterine cervix and in the endometrial adenocarcinomas with squamous cells. In most of these cases the tumors lacked ER and PgR although 80% of them contained the Mr 24,000 protein to a variable degree. It is suggested that Mr 24,000 protein is involved in growth and differentiation (the Mr 24,000 protein is a heat shock protein) and that the gene coding of this protein is under hormonal control only in those tissues where growth and differentiation are strongly hormonally controlled (breast and endometrium).

Recent evidence from our laboratory and from others suggested that pretreatment with alpha-difluoromethylornithine (DFMO) sensitizes some human and rodent tumor cell lines to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Many human tumor cells are resistant to chloroethylnitrosourea-induced DNA interstrand cross-linking and cell kill due to their high levels of the DNA repair protein O6-alkylguanine DNA alkyltransferase. We therefore investigated DFMO-mediated sensitization to BCNU in BCNU-sensitive and -resistant cells. Colony formation assays were used to compare BCNU cytotoxicity in DFMO-pretreated and control cultures of two colon tumor lines, HT-29 cells, which have high alkyltransferase levels and thus are BCNU-resistant, and BE cells, which are deficient in this repair capacity and thus are BCNU-sensitive. Polyamine depletion significantly enhanced BCNU cytotoxicity only for the repair-proficient HT-29 cell line. BE cells were 40-fold more sensitive to BCNU than were HT-29 cultures. However, in BE cells, no effect of polyamine depletion was found on cellular response to BCNU treatment at 72 h after DFMO treatment. Reverse-phase high-performance liquid chromatography assays of polyamine concentrations in cell extracts verified that DFMO produced comparable degrees of polyamine depletion for both cell lines. DNA alkaline elution analysis was used to monitor BCNU-induced formation of DNA single strand breaks, DNA interstrand cross-links, and DNA-protein cross-links. Equal concentrations of BCNU produced similar levels of strand breaks and DNA-protein cross-links in DFMO-pretreated and control cultures for both cell lines. These data suggest that DNA in polyamine-deficient HT-29 and BE cells is not more accessible to BCNU than is DNA in controls. No DNA interstrand cross-links were detected in either DFMO-pretreated or control HT-29 cells after BCNU treatment. Further, in BE cells which accumulate BCNU-induced DNA interstrand cross-links, no increase in the measureable levels of cross-links resulted from polyamine deficiency. Our observations suggest that mechanisms other than increased DNA interstrand cross-link formation may be mediating the enhanced efficacy of BCNU in polyamine-deficient HT-29 cell cultures. Our findings may also imply that cellular targets for BCNU other than DNA damage may be responsible for DFMO-induced chemosensitization in the repair-proficient cells.

The levels of milk-specific mRNAs (alpha-casein, whey acidic protein) and proteins (casein, alpha-lactalbumin) were examined in the DIM series of mammary preneoplastic outgrowth lines and tumors. The constitutive production of casein protein was variable among the preneoplastic DIM outgrowth lines maintained in virgin mice. Outgrowth line DIM-2 consistently expressed a very low level of casein mRNA and protein but no detectable alpha-lactalbumin protein. Outgrowth line DIM-4 expressed a high level of casein protein and no alpha-lactalbumin; however, by transplant generation 9, the levels of casein mRNA and protein were significantly decreased but remained greater than those found in DIM-2. Outgrowth line DIM-3 expressed high levels of casein protein during all transplant generations examined and sporadically exhibited detectable amounts of alpha-lactalbumin. The level of beta-casein mRNA in DIM-3 was 7 times greater than seen in DIM-4 outgrowths, but still only 2% of that measured in the normal mammary gland from lactating mice. The majority of tumors derived from the DIM outgrowth lines expressed very low levels of beta-casein mRNA and total casein protein, although occasionally tumors were observed with very high levels of casein expression. Immunoblot analysis of cellular extracts indicated that alpha, beta-, and gamma-caseins were expressed in the three outgrowth lines to varying degrees. Whey acidic protein mRNA attained barely detectable levels in the best of cases. Only outgrowth line DIM-3 responded to a normal lactogenic stimulus (lactating mouse) with significantly increased levels of milk-specific products. As determined by avidin-biotin peroxidase staining, the percentage of alveoli expressing beta-casein and alpha-lactalbumin proteins in lactating mice increased from 20 and 0%, respectively, in virgin mice to 85 and 40%, respectively, in lactating mice. Similarly, the levels of mRNA for beta-casein and whey acidic protein increased 8- and 5.5-fold, respectively. These results indicate that the cell populations selected by serial transplantation of preneoplastic mammary tissues fall into at least three categories with respect to expression of mammary-specific differentiation products: uninducible, inducible, constitutive. The DIM-3 outgrowth line appears to represent a highly inducible cell population. As concluded in earlier investigations, there was no correlation between secretory activity, morphology of the outgrowths, and tumor-producing capabilities in virgin mice. The effect of a normal lactogenic stimulus on the tumor potential of the DIM-3 line is currently under investigation.

The sensitivity of MXT mouse mammary tumors to ovarian hormones was assessed by the following parameters: growth in vivo; presence or absence of estrogen receptors and progesterone receptors; and histological differentiation. A spontaneous evolution from hormone sensitivity (HS) to hormone independence (HI) was observed when the tumors underwent monthly transplantation onto intact recipient mice, with the tumors fulfilling the criteria of HI tumors after the 12th transplantation. In contrast, the tumors recovered most of the criteria of hormone sensitivity when pituitary isografts were placed under the kidney capsules of HI tumor-bearing animals or when these animals received daily administrations of prolactin over several months. Sensitivity to 17 beta-estradiol, progesterone, or prolactin was further assessed by actinomycin binding on the nucleus and thymidine labeling index, both measured by autoradiography. These technical approaches revealed that 17 beta-estradiol and prolactin stimulated the thymidine labeling index of both HI (despite the lack of detectable estrogen receptors) and HS MXT tumors whereas progesterone influenced only that of HS cancers. The three hormones significantly stimulated [3H]actinomycin D binding within HS tumors but not within HI ones. However, such "HI" tumors were characterized by increased actinomycin binding and thymidine labeling index in comparison with HS neoplasms. Thus, all the data presently reported strongly suggest that prolactin is able to restore the hormone-sensitive phenotype within so-called MXT hormone-independent tumors.

Carbohydrate antigen (CA) 19-9 identified by a murine monoclonal antibody against a colorectal carcinoma antigen is thought to be a sialylated Lewis (Le)a blood group antigen and occurs in high concentration in serum of patients with pancreatic carcinoma. This study was designed to identify the relationship between Lewis antigens and CA 19-9 in patients with pancreatic cancer. The following analyses were performed in 20 pancreatic cancer patients: Lea and Leb antigen phenotype in saliva (modified enzyme-linked immunosorbent assay) or on red cells (hemagglutination); CA 19-9 levels (radioimmunoassay) in serum; and CA 19-9 and Lea and Leb expression (immunoperoxidase assay) on tumor tissue. Lea-b- patients based on salivary phenotype failed to express CA 19-9 in tumor tissue and had normal or low levels of CA 19-9 (less than 37 units/ml) in serum (P = 0.0011, versus Lea+b- and Lea-b+ patients). Eighty-eight % of Lea+b- and Lea-b+ patients had elevated serum CA 19-9 levels (greater than 37 units/ml). All Lea+b- and Lea-b+ patients expressed both Lea and Leb antigens in tumor tissue. These results support the view that Lea-b- pancreatic cancer patients cannot manufacture CA 19-9. Surprisingly, Lea-positive patients express Leb antigen in tumor tissue; in this subgroup, Leb antigen may be a tumor-specific biomarker.

The simultaneous quantitation of nuclear antigens and DNA content is presented using monoclonal antibodies and flow cytometric analysis, with paraffin-embedded human colonic pathology specimens utilized as source material. The monoclonal antibodies evaluated were shown by immunogold electron microscopy to recognize nuclear proteins preferentially associated with interchromatin (p105) and heterochromatin (p34) regions. Indirect immunofluorescence analysis of p105 revealed two distinct G1-G0 cell subpopulations in cells from normal colonic epithelium and colonic adenocarcinomas. In addition, enhanced levels of both p105 and p34 were observed in aneuploid DNA content stemlines, relative to diploid cells. Cell-sorting experiments performed on cells sorted on the basis of p105 and DNA contents reveal the capability of this method for identifying morphologically heterogeneous cell subpopulations. Other data suggest that p105 is differentially expressed in well-differentiated versus poorly differentiated tumor regions. The potential utility of this approach for the retrospective study of proliferation-associated antigens and protooncogene protein products is discussed.

Malignant changes are often accompanied by alterations in activity and composition of the plasminogen activators (PA). To study the relationship between PA expression and the development of colorectal cancer, we determined urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) activity in normal mucosa (n = 80), adenomatous polyps (n = 76), and adenocarcinomas (n = 71) of the colon. Tissues obtained from surgical resection or polypectomy were analyzed for t-PA and u-PA activity in a specific enzymatic assay using plasminogen, a chromogenic substrate, and selective quenching with monospecific antibodies to both activators. The plasminogen activator activities were found to be changed in adenocarcinomas as compared to normal mucosa. The relative contribution of u-PA (expressed as percentage of u-PA) was raised from 6 to 50% for, respectively, normal mucosa and adenocarcinoma. This change could be attributed to a 3-fold decrease in t-PA activity and a 5-fold increase in u-PA activity in the carcinomas. Adenomatous polyps as a group showed percentages of u-PA [20.2 +/- 1.3 (SE)] which were intermediate as well as significantly different (P less than 0.001) from those of normal mucosa and adenocarcinomas. This observation was strengthened by a gradual rise in the relative contribution of u-PA in four resection specimens containing both adenomatous polyps and adenocarcinomas. Zymography showed the presence of minor quantities of PA-PA inhibitor complexes in the tissue extracts studied. The present study shows that the sequence of normal mucosa-adenomatous polyp-adenocarcinoma in the colon is associated with a parallel change in plasminogen activator activity. Thus, change in the regulation of plasminogen activator activity is an early event in the development of colorectal cancer.

Evidence suggests that the anticancer agents etoposide (VP16-213) and teniposide (VM26) produce DNA breaks and cytotoxicity by interaction with type II topoisomerase. Therefore, levels of type II topoisomerase may influence sensitivity to VP16-213 and VM26. We have characterized four lung carcinoma-derived cell lines for natural sensitivity or resistance to VP16-213 and VM26. Included in this study were two small cell lung carcinoma lines (SW900 and SW1271), an adenocarcinoma line (A549), and a large cell carcinoma (H157). SW1271 was the most sensitive line with a median inhibitory concentration for cell proliferation of 0.5 microM for VM26 and 2.7 microM for VP16-213, and SW900 was the most resistant with median inhibitory concentration values of 2.0 and 16 microM, respectively. A549 and H157 cells were intermediate in sensitivity to these drugs. Alkaline elution techniques were used to study in vivo formation and repair of single and double strand DNA breaks. Single strand DNA breaks were observed in SW1271 cells exposed to as little as 10 nM VM26 or 100 nM VP16-213 for 1 h, whereas SW900 cells required exposure to 10-fold higher concentrations of VM26 or VP16-213 to produce similar results. Single strand DNA breaks predominated only in SW1271 and A549 cells and then, only at low drug concentrations, whereas the ratios between single and double strand DNA breaks decreased at higher drug concentrations. Plots of cytotoxicity versus single and double strand DNA breakage revealed that cytotoxicity produced by both drugs was more closely related to double strand DNA break formation in all four cell lines. DNA breaks appeared rapidly upon addition of drug, reaching plateaus in DNA breaks within 30 min, and repair of both single and double strand DNA breaks occurred rapidly with time to repair one-half of the DNA breaks of 20 to 60 min in all four cell lines upon removal of drug, arguing against repair as a mechanism for drug resistance. DNA breakage was also observed in nuclei isolated from SW900 and SW1271 cells in similar magnitude to that observed in the respective cells. Results indicate that DNA breakage plateaus may reflect a steady-state equilibrium established between the drug and its nuclear target, possibly type II topoisomerase, and suggest that natural resistance to VP16-213 and VM26 may be due to different enzyme levels in sensitive and naturally resistant cells.

Carcinoembryonic antigen-like substance, previously detected in large amounts in the medium from cultures of human prostatic epithelial cells, also is present in extracts of benign and malignant human prostate. By column chromatography, the prostate-derived carcinoembryonic antigen-like substance derived from cultured prostate is the same as that in tissue extracts and is distinctly different from colon-derived carcinoembryonic antigen. The molecular weight of prostate-derived carcinoembryonic antigen-like substance is estimated to be greater than 5 x 10(5). Prostate-derived carcinoembryonic antigen-like substance may be a prostate-specific substance.

The interaction between platelets and tumor cells plays an important role in the hematogenous spread of certain malignant cancers. We found that metastatic clones of murine colon adenocarcinoma 26 induced platelet aggregation in a membrane protein-dependent manner. Two monoclonal antibodies (mAbs) of IgG2a class were generated against a highly metastatic colon 26 clone, NL-17. These two mAbs, designated 8F11 and 20A11, showed a two-fold higher level of NL-17 binding than a low metastatic clone, NL-14, which possesses low platelet-aggregating ability. Both mAbs retarded platelet aggregation induced by NL-17. Western blot analysis showed that both mAbs recognized the same Mr 44,000 membrane protein as antigen under reducing conditions. Trypsin treatment of NL-17 diminished the ability of the cells to induce platelet-aggregation and resulted in a decrease in the reactivity of the cells to 8F11. These results suggest that the Mr 44,000 membrane protein recognized by the two mAbs is a platelet-aggregating factor of colon 26 cells.

We have found that reversible differentiation into the myoepithelial cells of a human salivary gland adenocarcinoma cell line (HSG) occurs in growth medium containing dibutyryl cAMP (dB-cAMP). In the current study, the relationship between intracellular cAMP levels and anchorage-dependent and -independent growth or ras oncogene of p21 levels was analyzed in the differentiation process toward myoepithelial cells of HSG cells cultured in the presence of dB-cAMP. Correlation between the concentrations of dB-cAMP and intracellular cAMP in the HSG cells was statistically significant. There was a significant inverse correlation between the concentrations of dB-cAMP and colony-forming ability of the cells in semisolid agar or on a plastic surface. We have found the expression of ras p21 protein in HSG cells. When HSG cells were cultured in the presence of dB-cAMP and were committed to differentiate into myoepithelial cells, it was shown by double-antibody labeling technique and/or immunoblotting that the committed cells expressed myosin with a concomitant decrease of ras p21 protein. Moreover, intracellular cAMP levels were found to be inversely associated with ras p21 content of the cells. These findings indicate that the intracellular cAMP levels regulate significantly cell proliferation and ras p21 expression in HSG cells.

The localization of three carbohydrate antigens, Lex, Ley, and sialylated Lex-i, which are closely related to stage-specific embryonic antigen 1, in the lung of developing human embryos was investigated using specific monoclonal antibodies. In the 38-day-old embryo, when the primitive lung bud has appeared and developed into two lung sacs, only Ley antigen was specifically positive in the proliferating cells in the terminal portion of lung bud. In the 50-53-day-old embryos, the future bronchi were actively developing from the bronchial buds. At this stage, the Ley antigen was maximally expressed and the Lex antigen appeared in the bud cells. In the lung of the 12-week-old embryo, buds for the future bronchioles were lined by simple cuboidal epithelial cells, which were strongly positive for Lex antigen, weakly positive for Ley antigen, and still completely negative for sialylated Lex-i antigen. Sialylated Lex-i antigen finally appeared in 18-week-old embryos, in the cells of the terminal buds for the future alveoli. At this stage, the Lex and Ley antigens were already beginning to disappear and were only weakly positive in cells of terminal buds. At 20 weeks, only sialylated Lex-i antigen was weakly detected in the cells in the terminal buds; after 8 months, all three antigens were essentially not detected in the respiratory cells in most of the embryos examined in this study. Formation of bronchial glands was detected at 18 weeks, where the developing gland cells were specifically positive for sialylated Lex-i antigen. Ciliation of the bronchial epithelial cells started at 12 weeks and propagated thereafter. The ciliation was accompanied by the reappearance of Ley and Lex antigen in the epithelial cells. These findings collectively indicated that the three antigens all have a physiological significance as stage-specific developmental antigens of the human lung; those antigens were specifically present in the bud cells at each important step of the morphogenesis of the human lung, such as cells in the lung buds, bronchial buds, and terminal buds for the formation of the alveolus, and cells differentiating into bronchial gland cells. The three antigens gradually disappear in the later stage of development along with the maturation process of the lung. Stage-specific embryonic antigen 1 and related antigens are known to be associated with various human cancers, including lung cancers. We suggest that the expression of these antigens in the lung cancer cells is the result of the retrodifferentiation of the cancer cells to the stages of immature embryonic lung cells.

Intracellular sodium, chlorine, and potassium concentrations (mmol/kg dry weight) were determined by electron probe X-ray microanalysis of individual epithelial cells in freeze-dried 2-micrometer sections of mouse mammary tissue which were cut at -30 degrees. A model system was utilized in order to compare elemental content of cells from normal pregnant mammary tissue and preneoplastic and neoplastic mammary tissues from female BALB/cCrlMed mice. Animals were killed by cervical dislocation, and tissue was rapidly frozen in liquid propane. Normal mammary glands were obtained from primiparous mice at 16 to 17 days of gestation. Tissue from the hyperplastic alveolar nodule line D1 was removed from donor mice 12 to 16 weeks after transplantation into the cleared mammary fat pad. All mammary adenocarcinomas, D1T, were primary tumors which developed in mice with transplants of nodule line D1. Data were collected from five animals (10 cells/animal) in each of the three groups. It was found that the electrolyte content of cells of preneoplastic tissue was the same as that of the normal mammary tissue but was significantly elevated in neoplastic tissue (162, 130, and 48% increases for sodium, chlorine, and potassium, respectively). Thus, an increase in electrolyte content seems to be associated with the transformation to a neoplastic state and not associated with conversion to the preneoplastic state.

Wheat germ agglutinin (WGA)-binding cellular glycoproteins produced by HT-29 human colon carcinoma and its variant cells established from liver metastases in nude mice after intrasplenic injection were analyzed by polyacrylamide gel electrophoresis. On 5.5% polyacrylamide gels five major sialoglycoproteins (approximate Mr 115,000, 145,000, 190,000, 450,000, and 740,000) reactive with WGA were common to the parental and metastatic sublines. There was an additional component of Mr approximately 900,000 that was prominent in cells established from liver metastases. Specific removal of sialic acid from the glycoproteins eliminated WGA binding, indicating that all the WGA-binding glycoproteins including the Mr 900,000 component were sialoglycoproteins. Smith degradation following mild acid hydrolysis resulted in formation of WGA-binding carbohydrate chains on Mr 115,000, 145,000, 190,000, and 900,000 components, but not on Mr 450,000 and 740,000 components, which indicated that these two sialoglycoproteins bore different oligosaccharides from the other sialoglycoproteins. The Mr 900,000 component was more prominent with HT-29 cells growing in nude mice than those growing in vitro. WGA binding to the Mr 900,000 component of metastasis-derived HT-29 cells growing in a nude mouse was higher than that of parental cells growing in nude mice. The expression in liver metastases derived from parental as well as metastatic cells was higher than the primary tumor growing in the spleen of the same mouse, indicating that the levels of Mr 900,000 sialoglycoprotein (SGP = 900) were regulated by intrinsic and environmental factors. The influence of organ microenvironmental factors was confirmed by analyzing sialoglycoproteins of HT-29 cells growing in the liver of a nude mouse following intrahepatic injection. Analyses of human colorectal carcinoma tissues and liver metastases revealed a polydisperse WGA-reactive high-molecular-weight component similar to that seen in tumors growing in nude mice. The mean value of WGA binding to high-molecular-weight glycoproteins in the primary tumors of stage B1 patients was smaller than that of all other primary tumors. Comparison of primary tumors with liver metastases from the same patients indicated that the level of SGP-900-like high-molecular-weight glycoproteins in metastases was not always higher than those in primary tumors.

We have studied the effects of two human pancreatic cancer and two human small cell lung cancer cell lines on clotting and platelet aggregation. Both pancreatic lines markedly shortened recalcification times and induced platelet aggregation. The lung cancer lines produced little shortening of recalcification times and no platelet aggregation. The clotting and aggregation activities of the pancreatic lines were further characterized. Recalcification times following the addition of cancer cell line material to plasmas deficient in factors VII and X were markedly prolonged, suggesting that the activity is due to tissue factor. Hirudin, an inhibitor of thrombin from the saliva of leeches, and rabbit polyclonal immunoglobulin G anti-bovine brain tissue factor inhibited both procoagulant and aggregation activities. Apyrase (an enzyme degrading ADP), diisopropylfluorophosphate (a serine protease inhibitor) and L-trans-epoxysuccinylleucylamido(4-guanidino)butane (a cysteine protease inhibitor) failed to inhibit these activities. Increasing concentrations of heparin inhibited platelet aggregation. Subcellular fractionation studies showed these activities to be localized to the plasma membrane. The association between mucin and the acceleration of clotting has been well described. The absence of mucin in electron micrographs of these pancreatic whole cells, membrane fractions, and shed microvesicles, as well as the failure of chaotropic agents (i.e., agents stripping material extrinsic to the cell membrane such as mucin) to abrogate this activity support these activities being intrinsic to the plasma membrane. These data strongly suggest that these activities are due to tissue factor which appears to be released as microvesicles in vitro. The release of tissue factor via microvesicles in vivo is one possible mechanism for the coagulopathy sometimes seen in patients with pancreatic carcinoma.

The influence of diabetes and insulin treatment on the phospholipid content of R3230AC mammary tumors, a hormonally responsive neoplasm, was studied. Diabetes was induced by administration of streptozotocin 3 days prior to tumor implantation. Protamine zinc insulin, 3 IU/rat twice daily, was administered to tumor-bearing rats for 3 days. Enzymatically dissociated tumor cells from diabetic animals showed significant increases in phosphatidyl choline, lysophosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, and phosphatidic acid, compared to controls. Diabetic animals treated with insulin displayed reductions in phosphatidyl choline, lysophosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, and phosphatidic acid to levels approximating those found in intact (control) animals. However, neither diabetes nor insulin treatment altered sphingomyelin levels. Mammary tumor cells from diabetic animals showed a 21% increase in DNA content compared to that in intact controls and treatment of diabetic animals with insulin lowered DNA level significantly. The responsiveness of both phospholipids and DNA content to changes in the insulin milieu of the host suggest that phospholipids may play an important role in mediating the effects of insulin on growth of R3230AC tumors.