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Introduction: Trismus after oral cancer is frequent, with scarce evidence of surgical release treatment in these patients., Objectives: The objective of this study is to determine the results of trismus release and free flap reconstruction after oral cancer treatment, establish immediate and long-term results, and detect factors that influence outcome., Materials and Methods: A retrospective study was performed. Preoperative, intraoperative, and follow-up interincisal distances were measured. The intraoperative, long-term gain, and postoperative loss were calculated. Analysis of the long-term results with the preoperative and intraoperative variables was performed., Results: Surgical release immediately increased the interincisal distance by 29.25 mm. Sixty-six percent of this gain was lost due to trismus recurrence, giving a long-term interincisal gain of 9.90 mm. Worse results were observed in patients with previous high-stage tumors, maxillectomies, skin resections, and previous radiotherapy. The were no significant differences in the results depending on the type of release or reconstruction performed, with the exception of the reconstruction with the sural flap, which presented worse results. Patients with trismus of ≤10 mm could have more range for improvement after this surgery., Conclusions: The results of this surgery are moderate in the long term due to high trismus recurrence in spite of aggressive treatment., (© 2023 Wiley Periodicals LLC.)

Nephron loss stimulates residual functioning nephrons to undergo compensatory growth. Excessive nephron growth may be a maladaptive response that sets the stage for progressive nephron damage, leading to kidney failure. To date, however, the mechanism of nephron growth remains incompletely understood. Our previous study revealed that class III phosphatidylinositol-3-kinase (Pik3c3) is activated in the remaining kidney after unilateral nephrectomy (UNX)-induced nephron loss, but previous studies failed to generate a Pik3c3 gene knockout animal model. Global Pik3c3 deletion results in embryonic lethality. Given that renal proximal tubule cells make up the bulk of the kidney and undergo the most prominent hypertrophic growth after UNX, in this study we used Cre-loxP-based approaches to demonstrate for the first time that tamoxifen-inducible SLC34a1 promoter-driven CreER T2 recombinase-mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to inhibit UNX- or amino acid-induced hypertrophic nephron growth. Furthermore, our mechanistic studies unveiled that the SLC34a1-CreER T2 recombinase-mediated Pik3c3 downregulation inhibited UNX- or amino acid-stimulated lysosomal localization and signaling activation of mechanistic target of rapamycin complex 1 (mTORC1) in the renal proximal tubules. Moreover, our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibited amino acid-stimulated mTORC1 signaling and blunted cellular growth in primary cultures of renal proximal tubule cells. Together, both our in vivo and in vitro experimental results indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1-S6K1-rpS6 signaling pathway., (© 2020 Wiley Periodicals LLC.)

Background and Objectives: This study aims to review our experience in trismus release followed by free flap reconstruction after radiotherapy in oral and oropharyngeal cancer, describe the results obtained in long-term follow-up and identify possible predictors of outcome., Methods: Patients' demographics, tumor characteristics and treatment where retrieved. Surgical release and reconstructive procedures were detailed. Interincisor distances (IIDs) where measured preoperatively (PO-IID), intraoperatively after release (IO-IID) and in the last follow-up (FU-IID). Potential predictors of outcome in terms of interincisor long-term gain (LT-gain) and postoperative loss (PO-loss) were analyzed., Results: Twenty-eight patients were included in our study. The mean LT-gain was 8.9 ± 7.0 mm, and the PO-loss was 22.14 ± 7.27 mm. Patients with a PO-IID of 10 mm or less had a significant higher LT-gain (P = 0.038). Predictors of worse outcome included having received a previous maxillectomy during tumor ablation (lower LT-gain, P = 0.035), and previous buccal mucosa resection (higher PO-loss, P = 0.044)., Conclusions: Trismus release and free flap reconstruction after radiotherapy in oral and oropharyngeal cancer patients seems to be associated with modest long term results and a high incidence of trismus recurrence, particularly in cases of prior buccal resections or maxillectomy. Patients should be adequately informed and carefully selected before indicating the procedure., (© 2017 Wiley Periodicals, Inc.)

The surface cells of corneal epithelium are regularly shed off and replaced by new cells that are derived from limbal epithelial stem cells (LESC). LESC are believed to reside in the basal layer of the limbal epithelium and are characterized with high expression levels of ΔNp63, a transcription factor (TF) which is believed to play roles in the regulation of LESC proliferation. In this study, we examined the transcriptional regulation of ΔNp63 in limbal epithelial cell. We employed DNA pull down assay followed by LC/MS analysis and cDNA microarray analysis to identify the TFs that were capable of binding to ΔNp63 promoter or were expressed at higher levels in limbus over cornea. The TFs thus selected were further examined for their in vivo ΔNp63 promoter binding by chromatin immunoprecipitation assay. We identified six putative TFs (PAX6, EGR1, CEBPB, JUN, ATF3, and ARID5B) through the aforementioned approaches. Among them, PAX6 and EGR1 were shown to promote the transcription of ΔNp63 and led to increased cell proliferation. In contrast, CEBPB and ATF3 appeared to exert little or no effect on ΔNp63 expression, however, their silencing suppressed cell proliferation. Although JUN exhibited low promoter-binding specificity, however, it affected ΔNp63 expression and limbal epithelial cell proliferation in ways similar to that of PAX6 and EGR1. Intriguingly, ARID5B was highly expressed in the limbal epithelial cell, however, its silencing by siRNA did not obviously affect the expression of ΔNp63, nor did it reduce cell proliferation of the limbal epithelial cell., (Copyright © 2012 Wiley Periodicals, Inc.)

Transplantation of cultivated human limbo-corneal epithelial (HLE) cells has been recognized as an effective stem cell (SC) therapy for treating corneal epithelial SC deficiency caused by burn or other diseases. With this technique, cryo-preserved human intact amniotic membrane (IAM) has been successfully used as a cell culture substrate and carrier, and is reported to preferentially preserve HLE stem/progenitor cells in vitro. However, little is known about what factors released by HLE cells are involved in the progenitor cell-preserving mechanism. Using proteomic method, we identified 13 proteins over-expressed by HLE cells cultured on IAM, which included heat shock protein 70-1 (Hsp70-1), Hsp-27, glutathione (GSH) S-transferase, annexin A2, galectin-7, and protein S100-A9. Increased Hsp70-1 expression was confirmed by Western blot and real-time PCR. The role of Hsp70-1 in promoting HLE cell survival was demonstrated by increased apoptosis index and increased cleaved poly ADP-ribose polymerase (CPARP) formation in hsp70-1-silenced, but not normal HLE cells after exposure to sublethal UVB irradiation or hydrogen peroxide. To understand the regulatory mechanism of Hsp70-1 expression in HLE cells, the role of transcription factor deltaNp63 (a well-recognized HLE stem cell; SC marker) was studied. We found that over-expression of deltaNp63α by plasmid vector induced a corresponding increase in Hsp70-1 protein production. Likewise, Hsp70-1 expression decreased in HLE cells after addition of deltaNp63α SiRNA. Immunoconfocal microscopy also revealed a paralleled expression of both proteins in corneal specimens. Thus, deltaNp63α-associated Hsp70-1 over-expression may promote HLE progenitor cell survival on IAM, possibly through the cytoprotective and anti-apoptotic effect of Hsp70-1., (Copyright © 2011 Wiley Periodicals, Inc.)

Loss of adipose tissue, primarily due to increased lipolysis but also to an impairment of adipogenesis, is a key feature of weight loss in cancer cachexia. Because of the myriad pathogenic signaling pathways essential for atrophy of adipose tissue, effective therapeutic agents for cachectic adipose loss are lacking and urgently needed. The authors evaluated the effects of YC-1 on adipogenesis of 3T3-L1 preadipocytes, TNF-α- and tumor-cell-induced lipolysis in 3T3-L1 adipocytes, and cachectic weight loss in colon-26 adenocarcinoma-bearing mice because YC-1 has been shown to possess versatile pharmacological actions, including anticancer activity. It was found that YC-1 promotes the differentiation of 3T3-L1 preadipocytes into adipocytes through activation of Akt and extracellular signal-regulated kinase (ERK) signaling pathways as well as activation of several adipogenic mediators, such as peroxisome proliferator-activated receptor γ (PPARγ), insulin receptor α (IRα), insulin receptor substrate-3 (IRS-3) and glucose transporter-4 (GLUT-4). In the in vitro lipolysis models, YC-1 attenuates TNF-α-induced lipolysis of adipocytes by antagonizing TNF-α-mediated activation of ERK and downregulation of perilipin (PLIN). It was also found that YC-1 inhibits colon-26 adenocarcinoma cell-induced lipolysis of 3T3-L1 adipocytes. Moreover, YC-1 effectively rescues cachectic weight loss in colon-26 adenocarcinoma-bearing mice by blocking lipolysis, involving insulin. Taken together the results show that YC-1 with its anticancer and anticachexia talents is highly worth developing as a novel agent for cancer therapy., (Copyright © 2011 UICC.)

Background and Objectives: Kenalog application onto surgical wounds has been used to control dynamic pain following Laser-Assisted-Uvulo-Palatoplasty (LAUP) with good results in our department. When the effect was sub-optimal, insufficient ointment over the superior-lateral corner of wounds were always noted. Fulfilling the coating led to optimal results, and preliminary trials showed a good outcome of dynamic pain control as well by treating the corners only. The corners were thus named "sweet spots." This study aimed to verify the efficacy of applying Kenalog on "sweet spots" instead of whole surgical wounds to mitigate dynamic pain due to LAUP., Study Design/materials and Methods: This is an outcomes research. Fifty-five subjects with primary snoring treated with LAUP were studied. By using Kenalog, local treatments were applied to sweet spots during the first postoperative week. A visual analogous scale was used to evaluate dynamic pain on the 1st, 2nd, 3rd, and 7th days after LAUP., Results: Once sweet spots were treated, there was instant alleviation of dynamic pain; the average improvement level was ≥80% (P < 0.001, power = 100%). The area of sweet spots, in contrast to the significant relief in dynamic pain, occupied only about 30% of denuded mucosa., Conclusions: Dynamic pain after LAUP could immediately be mitigated by local treatments upon sweet spots using topical regimens. The large-scale relief of dynamic pain in contrast to the small area of sweet spots implies a congregation of free nerve endings there. Our finding invites further studies to correlate the neuroanatomy of the soft palate and dynamic pain caused by LAUP., (Copyright © 2011 Wiley Periodicals, Inc.)

Astrocytes, the major glial population in the central nervous system (CNS), can secrete thrombospondin (TSP)-1 that plays the role in synaptogenesis and axonal sprouting during CNS development and tissue repair. However, little is known about the regulation of TSP-1 expression in astrocytes under oxidative stress condition. Here, a hypoxic mimetic reagent, cobalt chloride (CoCl(2)), was used to initiate hypoxia-induced oxidative stress in primary rat astrocytes. CoCl(2) at the concentration range of 0.1-0.5 mM was found to cause no significant cell death in primary rat astrocytes. However, CoCl(2) at 0.2-0.5 mM increased intracellular reactive oxygen species (ROS) levels and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression that is known as a hallmark for oxidative damage. We further found that TSP-1 mRNA expression in astrocytes was inhibited dose- and time-dependently by CoCl(2). TSP-1 mRNA levels were increased in CoCl(2)-exposed astrocytes in the presence of the inhibitors (U0126 and PD98059) of mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERK), when compared to that detected in the culture only exposed to CoCl(2). Moreover, the inhibition in TSP-1 mRNA expression by CoCl(2) was blocked by the addition of the potent antioxidant, N-acetylcysteine (NAC). Thus, we conclude that CoCl(2) inhibits TSP-1 mRNA expression in astrocytes via a ROS mechanism possibly involving MAPK/ERK. This inhibition may occur after CNS injury and impair the supportive function of astrocytes on neurite growth in the injured CNS tissues.

The transcription factor p63 belongs to the p53 protein family and plays an important role in epithelial development. Recent studies showed that p63 is over-expressed in some human squamous cell carcinomas of the head and neck, suggesting a role in carcinogenesis. The p63 gene contains two promoters and alternative promoter usage generates two groups of proteins with (TAp63) or without (ΔNp63) the transactivation domain. Although the roles of TAp63 in epithelial development have been described in numerous recent studies, the regulation of its expression has not been elucidated. In this study, we showed that the transcriptional activity of the TAp63 promoter and TAp63 protein level were both up-regulated by an increased c-jun activity in Hep3B human hepatocellular carcinoma cell. Moreover, the elevated TAp63 expression was coincided with an increased binding of c-jun to the TAp63 promoter. Point mutation of the sp1 binding site within the TAp63 promoter region attenuated the effect of c-jun on TAp63 expression. Knockdown of TAp63 expression by shRNA led to increased proliferation of Hep3B cell compared to that of the mock cell, suggesting a growth suppressive effect of TAp63., (© 2010 Wiley-Liss, Inc.)

In a previous report, we showed that the in vivo cytotoxic activity of the natural killer (NK) cells isolated from resveratrol-pretreated rats is significantly enhanced compared with that of the non-pretreated rats; however, the underlying mechanism remains unclear. In the present study, we use cultured NK92 cell line to examine the possible signaling pathways underlying the resveratrol-induced activation. Using cultured K562, HepG2, and A549 cells as targets, we show that resveratrol pretreatment increases NK cell cytotoxicity in a dose-dependent manner. The enhanced cytotoxic effect is accompanied by increases in JNK and ERK-1/2 MAP kinase activity and perforin expression. Moreover, the expression of NKG2D, an upstream signaling molecule of the MAP kinases pathway, is also enhanced. Resveratrol-enhanced perforin expression and cytotoxic activity are effectively inhibited by pretreatment with the inhibitors of JNK (SP600125), ERK-1/2 (PD98059), or by siRNAs against JNK-1 and ERK-2. However, the inhibitors or siRNA to p38 exhibits no effect. Since IL-2 has been shown to induce NKG2D expression and perforin release, we therefore, examined whether IL-2 and resveratrol act in parallel. We show that IL-2 also stimulates perforin expression, however, when treated together with resveratrol, they exhibit no additive effect. The results suggest that in NK92 cells, resveratrol may act via a similar or overlapping pathway as that of IL-2, to enhance perforin expression and cytotoxic activity. Data presented strongly indicate that resveratrol act via NKG2D-dependent JNK and ERK-1/2 pathways.

p63 belongs to a member of the tumor suppressor protein p53 family. Due to alternative promoter usage, two types of p63 proteins are produced. The DeltaNp63 isoform lacks the N-terminal transactivation domain and is thought to antagonize TAp63 and p53 in target gene regulation. DeltaNp63 has been found to be overexpressed in numerous human squamous cell carcinomas, including nasopharyngeal carcinoma (NPC). However, the role of DeltaNp63 overexpression in NPC pathogenesis has not been clear. In this study, we use a DeltaNp63 overexpressing human NPC cell line (NPC-076) to explore the possible roles of DeltaNp63 in cell proliferation and cell-cycle regulation. We found that the proliferation of NPC-076 cell is greatly suppressed when the overexpressed DeltaNp63 is silenced by specific DeltaNp63 siRNA. Further studies show that DeltaNp63 silencing results in the upregulation of CKIs, including p27(kip1) and p57(kip2) in both mRNA and protein levels. Cell-cycle analysis shows that DeltaNp63 silencing also results in an increased G1 phase cell and apoptotic cell population. Our findings indicate that DeltaNp63 plays important roles in the regulation of NPC-076 cell-cycle progression, and may play a role in the maintenance of NPC-076 tumor cell phenotype., ((c) 2008 Wiley-Liss, Inc.)

Overexpression of DeltaNp63 has been observed in a number of human cancers, suggesting a role for DeltaNp63 in carcinogenesis. In the present study, we show that inhibition of glycogen synthase kinase-3beta (GSK-3beta) by lithium chloride (LiCl) elicited a stimulatory effect on DeltaNp63 promoter activity in HEK 293T cells. Exposure to LiCl induced DeltaNp63 promoter activation as well as DeltaNp63 protein expression in the cells. The effect of GSK-3beta on DeltaNp63 expression was further confirmed by the use of two highly specific GSK-3beta inhibitors, SB216763 and SB415286. Further study showed the presence of a putative beta-catenin responsive element (beta-catenin-RE) in the DeltaNp63 promoter region, and the stimulation of DeltaNp63 promoter activity by GSK-3beta inhibitor is markedly abolished by mutation or deletion of the putative beta-catenin-RE. Data are also presented to show that beta-catenin acts together with Lef-1 to influence DeltaNp63 promoter activity and protein expression., ((c) 2008 Wiley-Liss, Inc.)

In spite of the strong evidence for the carcinogenic activity of cadmium and its related compounds, the underlying molecular mechanisms that lead to malignant transformation in cells exposed to cadmium remain unknown. Recently, Joseph et al. [J. Biol. Chem. 227:6131-6136, 2002] have identified, cloned, and characterized the mouse Translation Elongation Factor-1 delta sub-unit (TEF-1 delta, GenBank Accession Number AF304351) as a novel cadmium-responsive proto-oncogene. Presently, additional studies regarding the oncogenic potential of TEF-1 delta have been carried out. Transfection of NIH3T3 cells with the pcDNA3.1 expression vector containing the TEF-1 delta cDNA in the sense (5'-->3') orientation resulted in overexpression of the encoded 31 kDa protein. Transfection-mediated overexpression of TEF-1 delta protein resulted in transformation of the cells as evidenced from the appearance of transformed foci. Cotransfection of the cells with a mixture of plasmid DNA consisting of TEF-1 delta cDNA in the sense (5'-->3') and in the antisense (3'-->5') orientation resulted in significant inhibition of translation of the TEF-1 delta protein. Antisense TEF-1 delta mRNA-mediated inhibition of translation of TEF-1 delta protein, furthermore, resulted in inhibition of TEF-1 delta-mediated transformation of NIH3T3 cells as evidenced from the decrease in the number of transformed foci. These results further confirm that overexpression of TEF-1 delta is oncogenic and the antisense TEF-1 delta mRNA expression reverses its oncogenic potential., (Copyright 2002 Wiley-Liss, Inc.)

Although nickel compounds are known carcinogens, the underlying carcinogenic mechanisms are not fully understood. The objective of this research was to determine if the genotoxic lesions of DNA strand breaks and DNA-protein crosslinks are present in nickel-transformed BALB/c-3T3 cells, and to further elucidate the potential carcinogenesis of insoluble and soluble nickel compounds through telomerase activity in nickel-transformed BALB/c-3T3 cell lines. DNA strand breaks, DNA-protein crosslinks and telomerase activity were investigated by single cell gel electrophoresis (comet assay), (125)I-postlabelling techniques, and the TRAP-silver staining assay, respectively. Results showed that both DNA strand breaks and DNA-protein crosslinks were present in nickel-transformed BALB/c-3T3 cells. However, the highest levels of DNA strand breaks and DNA-protein crosslinks were found in insoluble crystalline NiS-transformed cells and high levels of DNA strand breaks and DNA-protein crosslinks were also found in the transformed cells induced by two water-soluble NiCl(2) and NiSO(4) at moderate concentrations of cytotoxicity. These data suggest that these two genetic endpoints are useful biomarkers and are associated with cell transformation and carcinogensis of insoluble and soluble nickel compounds. Also, we found that the crystalline NiS- and NiCl(2)-transformed cells possessed a high telomerase activity. A weak telomerase was found in NiSO(4)-transformed cells. The results seem to indicate that in addition to crystalline NiS, some water-soluble nickel compounds such as NiCl(2) are also highly carcinogenic. These results may partly explain the cell transformation and relative carcinogenic potency of insoluble crystalline NiS, soluble NiCl(2), and NiSO(4)., (Copyright 2001 Wiley-Liss, Inc.)

It has been shown that cAMP may perturb the polypeptide growth factor-induced nuclear events. However, the possible interactions of the cAMP-protein kinase A (cAMP-PKA) and receptor tyrosine kinase pathways in the cytosol have not been fully elucidated. In this study, we use human astrocytoma cells as a model to investigate this issue. The results show that platelet-derived growth factor (PDGF)-induced receptor autophosphorylation in human astrocytoma cells is suppressed by dibutyryl-cAMP pretreatment and such suppression is not due to changes in the ligand-receptor binding properties. Further studies show that PDGF-induced tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and phosphatidylinositol 3-kinase (PI 3-kinase) are also suppressed in dibutyryl-cAMP-pretreated cells. The suppression of PLC-gamma 1 tyrosine phosphorylation was accompanied by a decreased production of water soluble inositol phosphates. In contrast, similar treatment with normal human astrocytes potentiates the tyrosine phosphorylation of PLC-gamma 1 and PI 3-kinase. The results indicate that cAMP can either negatively or positively modulate the PDGF receptor tyrosine kinase activity depending on the cell types examined.

CG-1 human nasopharyngeal carcinoma cells in monolayer culture formed both cohesive, epithelial-like colonies and scattered, fibroblastic-like colonies in mixed proportions. In the presence of exogenously added bFGF (4 ng/ml), about 85% of the colonies formed were fibroblastic-like. CG-1 cells were capable of synthesizing and releasing bFGF, and, when compared by the immunological method, cells in fibroblastic-lke colonies were found to contain higher levels of endogenous bFGF than cells in the epithelial-like colonies. Furthermore, cells in the peripheral region of the epithelial-like colonies, which were fibroblastic-like in morphology, also appeared to contain higher levels of endogenous bFGF. In addition, in the presence of suramin, neutralizing antibody to bFGF, or neutralizing antibodies to bFGF and EGF, the number of cohesive colonies formed was greatly increased. Moreover, addition of the 2 M NaCl-eluted heparin-Sepharose fraction of the CG-1 cell-coditioned medium promoted the formation of dispersed colony in a dose-dependent manner. The results suggest that bFGF can regulate CG-1 cell phenotype in an autocrine manner.

Chlorophyllin, a derivative of chlorophyll, is known to be an antimutagenic agent. Studies were performed to determine whether chlorophyllin can also inhibit morphological transformation of BALB/3T3 cells induced by carcinogens and complex mixtures. Chlorophyllin was added to the cultures simultaneously with carcinogens or complex mixtures while the transformation assay was conducted. At concentrations that did not significantly affect cell growth, chlorophyllin was found to inhibit morphological transformation induced by N-methyl-N'-nitro-N-nitrosoguanidine, 3-methylcholanthrene, 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, aflatoxin B1, and extracts of coal dust, tobacco snuff, and chewing tobacco. In all cases, the mean number of transformed foci per flask treated with chlorophyllin was significantly lower than that of untreated cultures. The reduction in the number of transformed foci was dependent on the concentration of chlorophyllin tested. These results indicate that chlorophyllin is an antitransforming agent.

The growth of a recently established human nasopharyngeal carcinoma cell line, CG-1, and 5 randomly selected, single-cell-derived sub-lines in serum-free medium and in fetal-bovine-serum(FBS)-containing medium was investigated. In basal medium supplemented with insulin, transferrin, fibronectin and high-density lipoprotein, cell growth was moderately stimulated by aFGF and EGF in a dose-dependent manner. In contrast, in medium containing as little as 0.5% FBS, most of the stimulatory effect of the aforementioned growth factors observed was masked. Western blotting analysis of the cell lysates and conditioned media showed that CG-1 and sub-line cells were all capable of synthesizing and releasing aFGF- and EGF-immunoreactive proteins. The amounts of these 2 growth factors synthesized and released appeared to vary among the parental cell line and sub-line cells. Moreover, the rate of basal proliferation of these cells appeared to be positively correlated with the amounts of aFGF- and EGF-immunoreactive proteins produced. Addition of the neutralizing antibodies to aFGF and EGF exerted a dose-dependent suppression on cell growth in medium containing 0.5% FBS. The results suggest a role of aFGF and EGF in autocrine growth stimulation of CG-1 and sub-line cells, and may explain the moderate response of these cells to exogenously added aFGF and EGF.

The contraction of collagen lattices made with foreskin fibroblasts in medium containing 1% fetal bovine serum was inhibited by intracellular cyclic AMP-raising drugs including cholera toxin (CT), forskolin, and dibutyryl-cAMP. The inhibition by CT was attenuated by insulin, acidic fibroblast growth factor (aFGF), and transforming growth factor-beta (TGF-beta). All three peptide factors have previously been reported to promote collagen lattice contraction by arterial smooth muscle cells and/or fibroblasts. Incubation of cells suspended in collagen gels with CT and forskolin resulted in a transient rise of the intracellular cyclic AMP levels, which peaked at 2 hr and 30 min, respectively, after drug exposure. Cholera toxin-induced intracellular cyclic AMP increase was attenuated by TGF-beta, but not by aFGF and insulin, when added simultaneously. Thus, TGF-beta may attenuate CT's inhibition on collagen lattice contraction by attenuating CT-induced intracellular cyclic AMP increase, whereas the attenuation by insulin and aFGF on the inhibition of lattice contraction may be mediated by a cyclic AMP-independent mechanism.

Transplacental cytogenetic effects of triethylenemelamine (TEM), benzene, and vinblastine on maternal mice and their fetuses have been investigated using micronucleus and sister chromatid exchange (SCE) as genetic endpoints. CD-1 mice were treated on day 14 and 15 of gestation with TEM (0.125, 0.25, and 0.5 mg/kg), benzene (439,878, and 1,318 mg/kg), and vinblastine (0.5, 1, and 2 mg/kg) by intraperitoneal injection at 24 hr intervals, and sacrificed 40 hr after the first injection. Erythrocytic precursor cells in maternal bone marrow and fetal livers (2-4) from each pregnant mouse were used for the micronucleus and/or the SCE analyses. Significant dose-related increases in both micronuclei and SCE were found in maternal bone marrow and fetal liver following TEM treatment. Benzene at the highest dose (1,318 mg/kg) also caused a significant increase in micronuclei and SCE in both maternal bone marrow and fetal liver cells. The embryonic genotoxic effect of TEM was much higher than that of benzene for both genetic endpoints, and the frequency of micronuclei induced by benzene was higher in fetal liver than in maternal bone marrow cells. Vinblastine, a spindle poison, induced micronuclei but not SCE. Micronuclei induction by vinblastine was 7 fold greater in maternal bone marrow than in fetal liver cells. All three chemicals were cytotoxic in maternal bone marrow cells, but not in fetal liver cells except for TEM, which showed a weak cytotoxicity in fetal liver cells in the micronucleus assay. These results indicate that TEM, benzene, and vinblastine are transplacental genotoxicants in mice.

The contraction of collagen lattices made with arterial smooth muscle cells was studied in medium MCDB 107 without serum or supplemented with 1% fetal bovine serum, plus insulin, transferrin, and low-density lipoprotein. Under these conditions, smooth muscle cell mitogens including HBGF-1 (aFGF), PDGF, and EGF stimulated contraction. Stimulation by HBGF-1 was more profound than with other factors tested. HBGF-1 stimulation of lattice contraction was blocked by protein synthesis inhibitors, but not inhibitors of DNA synthesis. Histological observations indicated that HBGF-1 also enhanced the maintenance of healthy cells in the lattice. Taken together, these observations suggest that HBGF-1 stimulates lattice contraction, not by a mitogenic effect, but by stimulating synthesis of specific cellular proteins. Since the greatest effects of HBGF-1 on lattice contraction were seen during the first 72 h following casting, the effects on maintenance of cell viability are probably less important in promoting lattice contraction.

Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and heparin-binding growth factor-1 (HBGF-1) stimulated the proliferation of a variant of the human osteosarcoma cell line, MG-63-LS (LS = low serum). Transforming growth factor beta (TGF-beta) completely inhibited cell growth in basal medium supplemented with 2% fetal calf serum (FCS), blocked PDGF- and EGF-stimulated cell proliferation, and modulated that of HBGF-1. PDGF, but not EGF or HBGF-1, activated the inositol trisphosphate/diacylglycerol (IP3/DAG) second message system in a dose-dependent manner. EGF inhibited phosphoinositol lipid turnover and HBGF-1 and TGF-beta stimulated phosphatidylinositol hydrolysis to produce inositol phosphate (IP) but not IP3. Preincubation of quiescent cells with TGF-beta for 30-40 minutes prior to the addition of PDGF resulted in an inhibition of PDGF-induced production of IP3. This suggested that TGF-beta was an indirect inhibitor and blocked PDGF-stimulated cell growth in part by interfering with the generation of the second messenger, IP3.

The growth of MG63 human osteosarcoma cell line in 5% serum is stimulated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or heparin-binding growth factor-1 (HBGF-1). The mitogenic effect of EGF and PDGF is completely blocked by TFG-beta at 1 ng per ml and the effect of HBGF-1 is attenuated by 75-80%. Treatment of MG63 cells with TGF-beta reduces HBGF-1 receptor binding affinity from 1.24 x 10(-11) M to 3.51 x 10(-11) M with no change on the receptor number (1.1 x 10(3) per cell). The receptor-binding affinity of EGF and PDGF is not altered by TGF-beta treatment; however, the number of EGF receptor is increased by 25%. Both EGF and PDGF stimulate MG63 cellular tyrosine kinase activity, and such stimulation is inhibited by TGF-beta pretreatment. No change in the cellular protein tyrosine phosphorylation pattern can be detected in HBGF-1-stimulated cells with and without TGF-beta pretreatment. These data suggest that TGF-beta inhibits EGF and PDGF mitogenicity by blocking EGF- and PDGF-stimulated tyrosine kinase activity and attenuates HBGF-1 mitogenicity by decreasing its receptor affinity.

Recently improved culture conditions for human adult arterial endothelial and smooth muscle cells from a wide variety of donors have been used to study the effects of lipoproteins on proliferation of both cell types in low serum culture medium. Optimal growth of endothelial and smooth muscle cells in an optimal nutrient medium (MCDB 107) containing epidermal growth factor, a partially purified fraction from bovine brain, and 1% (v/v) lipoprotein-deficient serum was dependent on either high- or low-density lipoprotein. High- and low-density lipoprotein stimulated cell growth by three- and five-fold, respectively, over a 6-day period. Optimal stimulation of both endothelial and smooth muscle cell growth occurred between 20 and 60 micrograms/ml of high- and low-density lipoproteins, respectively. No correlation between the activation of 3-hydroxyl-3-methylglutaryl coenzyme. A reductase activity and lipoprotein-stimulated cell proliferation was observed. Lipid-free total apolipoproteins or apolipoprotein C peptides from high-density lipoprotein were partially effective and together with oleic acid effectively replaced native high-density lipoprotein for the support of endothelial cell growth. In contrast, apolipoproteins or apolipoprotein C peptides from high-density lipoprotein alone or with oleic acid had no effect on smooth muscle cell proliferation. The results suggest a functional role of high- and low-density lipoproteins and apolipoproteins in the proliferation of human adult endothelial and smooth muscle cells.

We investigated the effects of three serine protease inhibitors (leupeptin, soybean trypsin inhibitor, and aprotinin) on the serum-free growth of two transformed cell lines in soft agar. Aprotinin markedly enhanced the growth of rat embryo fibroblasts that had been transformed by polyoma middle T antigen (PyMLV-REF52), while having only a slight effect on the colonial growth of SV40 transformed Balb/c 3T3 cells (SV3T3-Aga). Leupeptin and soybean trypsin inhibitor, on the other hand, significantly enhanced the growth of SV3T3-Aga cells while having little effect on PyMLV-REF52 growth. We observed no stimulatory effect of any of the protease inhibitors on serum-free monolayer growth. Under conditions of excess aprotinin, PyMLV-REF52 cells were found to be unresponsive to epidermal growth factor (EGF) at a concentration that would normally stimulate agar colony growth. However, aprotinin was not capable of supporting colony formation with transforming growth factor-beta. These results indicate that aprotinin acts primarily as a protease inhibitor in spite of its structural homology to EGF and that EGF may promote the soft agar growth of these cell lines either by inhibiting proteolysis directly or by enhancing the synthesis of a serine protease inhibitor.

The lipid-free apolipoproteins of human high density lipoprotein (HDL) have been assayed for their ability to substitute for native HDL in promoting the growth of a SV40-transformed REF52 cell line in serum-free medium. Total HDL-apolipoproteins (apoHDL) were found to mimic almost exactly the growth promoting effects of whole HDL. The apoHDL-associated growth promoting activity eluted from a Sephacryl S-200 column in two separate fractions coinciding with the protein peaks of apolipoprotein A-I and the C group of apolipoproteins. These two fractions, designated S-II and S-IV, respectively, acted additively in promoting WT1A cell growth when tested at saturating concentrations. The active component in the S-II fraction maximally stimulated WT1A cell growth at 40-60 micrograms/ml and was identified as apolipoprotein A-1 by NaDodSO4 polyacrylamide gel electrophoresis and affinity chromatography on anti-(apoA-I). The active component in the S-IV fraction was maximally active at 1-2 micrograms/ml and was identified as apolipoprotein C-III by DEAE ion exchange high pressure liquid chromatography and polyacrylamide gel electrophoresis (at pH 8.3) in 6 M urea. These results indicate that the growth promoting effect of HDL on WT1A cells is mediated via the HDL-apolipoproteins, A-I and C-III, and that the mechanism responsible does not necessarily involve their participation in the uptake (or utilization) of HDL-associated lipids.