High Rate Of DNA Synthesis Research Articles

Sodium arsenite alters cell cycle and MTHFR, MT1/2, and c-Myc protein levels in MCF-7 cells

There is limited available information on the effects of arsenic on enzymes participating in the folate cycle. Therefore, our aim was to evaluate the effects of sodium arsenite on the protein levels of methylenetetrahydrofolate reductase (MTHFR) and dihydrofolate reductase (DHFR) and its further relationship with the expression MT1/2 and c- myc in MCF-7 cells. Arsenite treatment (0–10 μM) for 4 h decreased MTHFR levels in a concentration-dependent fashion without significant effects on DHFR. The effects on MTHFR were observed at arsenite concentrations not significantly affecting cell viability. We also observed an increase in S-phase recruitment at all concentrations probed. Lower concentrations (< 5 μM) induced cell proliferation, showing a high proportion of BrdU-stained cells, indicating a higher DNA synthesis rate. However, higher concentrations (≥ 5 μM) or longer treatment periods induced apoptosis. Arsenite also induced dose-dependent increases in MT1/2 and c-Myc protein levels. The levels of MTHFR were inversely correlated to MT1/2 and c-Myc overexpression and increased S-phase recruitment. Our findings indicate that breast epithelial cells are responsive to arsenite and suggest that exposure may pose a risk for breast cancer. The reductions in MTHFR protein levels contribute to understand the mechanisms underlying the induction of genes influencing growth regulation, such as c- myc and MT1/2. However, further research is needed to ascertain if the effects here reported following short-time and high-dose exposure are relevant for human populations chronically exposed to low arsenic concentrations.

MCT-1 Oncogene Contributes to Increased In vivo Tumorigenicity of MCF7 Cells by Promotion of Angiogenesis and Inhibition of Apoptosis

Overexpression of a novel oncogene MCT-1 (multiple copies in a T cell malignancy) causes malignant transformation of murine fibroblasts. To establish its role in the pathogenesis of breast cancer in humans, we generated stable transfectants of MCF7 breast cancer cells negative for endogenous MCT-1 (MCF7-MCT-1). Overexpression of MCT-1 in these cells resulted in a slight elevation of estrogen receptor-alpha, and higher rates of DNA synthesis and growth in response to estradiol compared with the empty vector control (MCF7-EV). The pure antiestrogen fulvestrant inhibited the estradiol-stimulated proliferation of MCF7-MCT-1 cells. The MCF7-MCT-1 clones showed increased invasiveness in the presence of 50% serum compared with the MCF7-EV. In a tumor xenograft model, MCT-1-overexpressing cells showed higher take rates and formed significantly larger tumors than MCF7-EV controls. When we examined angiogenic phenotype and molecular mediators of angiogenesis in MCF7-MCT-1 tumors in vivo, we found greater microvascular density and lower apoptosis in the MCF7-MCT-1 tumors compared with MCF7-EV controls accompanied by a dramatic decline in the levels of angiogenesis inhibitor, thrombospondin-1 (TSP1). In vitro, blocking TSP1 in the medium conditioned by MCT-1-negative cells restored its angiogenic potential to that of the MCF7-MCT-1 cells. Conversely, despite an increase in mRNA encoding vascular endothelial growth factor upon MCT-1 overexpression, vascular endothelial growth factor protein levels have not been notably altered. Taken together, our results suggest that MCT-1 may contribute to the pathogenesis and progression of human breast cancer via at least two routes: promotion of angiogenesis through the decline of TSP1 and inhibition of apoptosis.

Benzoquinone activates the ERK/MAPK signaling pathway via ROS production in HL-60 cells

Benzene (BZ) is a class I carcinogen and its oxidation to reactive intermediates is a prerequisite of hematoxicity and myelotoxicity. The generated metabolites include hydroquinone, which is further oxidized to the highly reactive 1,4-benzoquinone (BQ) in bone marrow. Therefore, we explored the mechanisms underlying BQ-induced HL-60 cell proliferation by studying the role of BQ-induced reactive oxygen species (ROS) in the activation of the ERK–MAPK signaling pathway. BQ treatment (0.01–30 μM) showed that doses below 10 μM did not significantly reduce viability. ROS production after 3 μM BQ treatment increased threefold; however, catalase addition reduced ROS generation to basal levels. FACS analysis showed that BQ induced a fivefold increase in the proportion of cells in S-phase. We also observed a high proportion of Bromodeoxyuridine (BrdU) stained cells, indicating a higher DNA synthesis rate. BQ also produced rapid and prolonged phosphorylation of ERK1/2 proteins. Simultaneous treatment with catalase or PD98059, a potent MEK protein inhibitor, reduced cell recruitment into the S-phase and also abolished the ERK1/2 protein phosphorylation induced by BQ, suggesting that MEK/ERK is an important pathway involved in BQ-induced ROS mediated proliferation. The prolonged activation of ERK1/2 contributes to explain the increased S-phase cell recruitment and to understand the leukemogenic processes associated with exposure to benzene metabolites. Thus, the possible mechanism by which BQ induce HL-60 cells to enter the cell cycle and proliferate is linked to ROS production and its growth promoting effects by specific activation of regulating genes known to be activated by redox mechanisms.

Dedifferentiation and proliferation of surviving epithelial cells in acute renal failure.

In contrast to the heart or brain, the kidney can completely recover from an ischemic or toxic insult that results in cell death. During recovery from ischemia/reperfusion injury, surviving tubular epithelial cells dedifferentiate and proliferate, eventually replacing the irreversibly injured tubular epithelial cells and restoring tubular integrity. Repair of the kidney parallels kidney organogenesis in the high rate of DNA synthesis and apoptosis and in patterns of gene expression. As has been shown by proliferating cell nuclear antigen and 5-bromo 2'-deoxyuridine labeling studies and, in unpublished studies, by counting mitotic spindles identified by labeling with antitubulin antibody, the proliferative response is rapid and extensive, involving many of the remaining cells of the proximal tubule. This extensive proliferative capacity is interpreted to reflect the intrinsic ability of the surviving epithelial cell to adapt to the loss of adjacent cells by dedifferentiating and proliferating. Adhesion molecules likely play important roles in the regulation of renal epithelial cell migration, proliferation, and differentiation, as do cytokines and chemokines. Better understanding of all of the characteristics resulting in dedifferentiation and proliferation of the proximal tubule epithelial cell and cell-cell and cell-matrix interactions important for this repair function will lead to novel approaches to therapies designed to facilitate the processes of recovery in humans.

P2 receptors on macroglial cells: Functional implications for gliosis

AbstractThe present review summarizes data about the expression of P2 receptors in two types of macroglial cells, i.e., in astrocytes of the nucleus accumbens (NAc) and Müller glial cells of the retina. Changes in the expression of these receptors during pathological conditions may indicate an involvement in the induction and/or maintenance of reactive astrogliosis. In fact, astrocytic proliferation was induced in the NAc by the application of different P2X and P2Y receptor agonists, such as 2‐methylthio ATP (2‐MeSATP), α,β‐methylene adenosine 5′‐triphosphate (α,β‐meATP) and adenosine 5′‐O‐(2‐thiodiphosphate (ADP‐β‐S). An increased content of glial fibrillary acidic protein, astrocytic hypertrophy, and proliferation as indicated by enhanced incorporation of 5‐bromo‐2′‐deoxyuridine (BrdU) was found under these conditions; an enhanced BrdU incorporation signalizes a higher rate of DNA synthesis. Müller glial cells of the human retina express both metabotropic P2Y and ionotropic P2X7 receptors. Application of the P2X7 receptor agonist benzoyl‐benzoyl ATP (BzATP) evoked a cationic conductance increase that led to the depolarization of cells and to Ca2+ entry from the extracellular space. Long‐lasting application of BzATP, however, did not cause the formation of pores permeable for large molecules, excluding the presence of classic P2X7 receptors. Müller glial cells of the human retina show increased P2X7 receptor‐mediated cationic currents during proliferative gliosis. A role of P2X7 receptors in glial proliferation is also indicated by the observation that the DNA synthesis rate of cultured Müller cells is enhanced in the presence of BzATP, as shown by the measurement of BrdU incorporation. The data indicate that activation of P2 receptors may exert both short‐ and long‐term effects in macroglial cells, and that these receptors may be involved in glial responses to pathological conditions. Drug Dev. Res. 53:140–147, 2001. © 2001 Wiley‐Liss, Inc.

Overexpression of interleukin-6 in human basal cell carcinoma cell lines increases anti-apoptotic activity and tumorigenic potency

Interleukin-6 (IL-6) is a pleiotropic cytokine that is capable of modulating the diverse functions of cells such as acute phase responses and inflammation. Excessive or insufficient production of IL-6 may contribute to certain diseases of the skin. The aim of this study was to investigate the possible role of IL-6 in the tumorigenesis of basal cell carcinoma (BCC). Initially, we transfected IL-6 expression vector, under the control of a CMV promoter, into human BCC cells and successfully obtained IL-6-overexpressing clones (BCC/IL-6-c1 and BCC/IL-6-c2) and a mixture (BCC/IL-6). DNA synthesis assay determined using (3)H-thymidine pulse incorporation revealed that IL-6-expressing BCC cells exhibited a much higher DNA synthesis rate than the neo control or parental BCC cells. We also detected a greater abundance of IL-6-expressing cell colonies formed in soft agar than in the vector control cells. Furthermore, BCC/IL-6 cells, but not vector control cells, were resistant to UV and photodynamic therapy (PDT)-induced apoptosis, as confirmed using DNA fragmentation and morphologic change analyses. Immunoblot analysis showed that Mcl-1, an anti-apoptotic protein, was specifically up-regulated IL-6 transfectants but not in the control cells. Transient transfection of IL-6 transfectants with antisense mcl-1 greatly enhanced their apoptosis frequency by UV treatment. In tumorigenesis assay, IL-6 transfected clones formed tumors in nude mice more rapidly than the control cells. These tumors appeared to be highly vascularized using pathological examination. Supportive of this finding, we found that IL-6 transfected cells expressed elevated levels of two angiogenic factors, cyclooxygenase (Cox)-2 and vascular endothelial growth factor (VEGF). These results suggest that overexpression of IL-6 enhances the tumorigenic activity of BCC cells by both suppressing apoptosis and actively promoting angiogenesis.

In vitro DNA synthesis in freshly separated human breast cancer cells assessed by tritiated thymidine incorporation assay: relationship to the long-term outcome of patients.

Tumour growth rate has a significant effect on the clinical course of various malignancies. The present study was designed to assess whether in vitro DNA synthesis in freshly separated breast cancer cells is a useful marker in evaluating growth rates and in predicting the clinical outcome of patients. From 1982 to 1992, DNA synthesis was assessed by [3H]thymidine incorporation in 97 samples of primary lesions from 94 patients with breast cancer. The patients were followed for 5-15 years and their outcome was surveyed in January 1998. The level of DNA synthesis did not correlate with the patients' age, clinical stage or expression of oestrogen receptor. However, it correlated significantly with the histological grade. In 89 patients, whose outcome was reported, the survival rate in the group with a high rate of DNA synthesis (log10c.p.m. 3.0 or more) was significantly lower than that in the low-level group; the 5- and 10-year survival rates were 84 and 74 per cent for the low synthesis group (n = 46), and 60 and 46 per cent for the high synthesis group (n = 43) respectively. This was also noted in patients with stage 1 or 2 cancers, for whom the 5- and 10-year survival rates were 100 and 90 per cent for the low synthesis group (n = 25), and 75 and 70 per cent for the high synthesis group (n= 35). Multivariate analysis supported this significant correlation for DNA synthesis in the prognosis of patients after mastectomy. Furthermore, the level of DNA synthesis was significantly higher in 18 patients who died from a recurrence within 3 years after operation than in 56 survivors, and the level of DNA synthesis also correlated significantly with the survival period in the 33 patients who died. The level of DNA synthesis in breast cancer was variable, and was independent of the clinical stage or oestrogen receptor status. However, a high level of DNA synthesis was a positive indicator of a high risk of recurrence after operation, especially for stage 1 or 2 breast cancer. In vitro DNA synthesis may account for some of the clinical characteristics of breast cancers.

Plant cell-directed control of virion sense gene expression in wheat dwarf virus.

We have used particle bombardment (biolistics) to deliver replication-competent wheat dwarf virus (WDV)-based constructs, carrying reporter gene sequences fused to the virion sense promoter (Pv) or the CaMV 35S promoter, to suspension culture cells and immature zygotic embryos of wheat. While the replication of WDV double-stranded DNA forms (replicons) was equivalent between wheat suspension culture cells and embryos, GUS reporter gene activity was 20-40 times higher in the embryo cultures. Maximum expression of WDV replicons occurred in the embryonic axis tissue of wheat embryos but their expression in suspension cells was compromised, compared with transiently maintained input plasmid DNA containing the same sequences. From these studies, we propose that WDV replicons are subject to a host cell-controlled competency for virion sense transcription. The term competency is used to distinguish between the phenomenon described here and control of gene expression by specific transcription factors. Control of competency is independent of Pv, the replacement 35S promoter and of the complementary sense control of virion sense expression involving specific sequences in Pv. We propose that factors controlling the competency for replicon expression may be present in cells which, as well as maintaining high rates of DNA synthesis, are totipotent. Cell type control of active chromatin, methylation of specific sequences in WDV minichromosomes and/or interaction of virus-encoded proteins with specific host factors are considered as possible mechanisms.

Release of clonal block in B cell chronic lymphocytic leukaemia by engagement of co-operative epitopes on CD40

The clonal cells of patients with B-chronic lymphocytic leukaemia (B-CLL)—which essentially reside in a resting configuration—are characterised by a relative refractoriness to the normal signals for B cell growth and differentiation. Previously it has been shown that, using an in vitro culture system where CD40 is hyper-crosslinked by monoclonal antibody (mAb) held on CD32-transfected mouse L cells, the clonal block in B-CLL cells can be released with a resultant high rate of DNA synthesis ensuing. In the present study, we report that such release can be achieved purely with soluble reagents whereby co-operative epitopes on CD40 are targeted by the combined use of mAb and soluble recombinant CD40L. Substantial levels of DNA synthesis were induced under such conditions in 7 18 patients using CD40-targeted reagents alone and in 16 18 patients in the additional presence of interleukin 4. Possible extrapolation of these findings to novel therapeutic modalities could be envisaged.

Maturation and function of human fetal pancreatic cells after cryopreservation.

If transplantation of endocrine tissue is to become a therapy for a significant number of insulin-dependent diabetic individuals, tissue sources other than adult human islets will be required. Because human fetal pancreatic cells may offer that alternative, we have tried to find the optimal combination of tissue culture and cryopreservation methods for use in transplantation. Islet-like cell clusters (ICCs), cryopreserved according to reported methods for adult human islets, survived poorly after thawing. In contrast, the yield of ICCs was comparable after collagenase digestion of fresh and cryopreserved pancreatic fragments. However, the ICCs derived from cryopreserved tissue contained a higher proportion of nonepithelial cells, and the recovery of insulin was only 28%, as compared with freshly cultured cells. These ICCs had a lower DNA content and a higher rate of DNA synthesis. Moreover, cryopreserved cells released a higher fraction of their insulin content in basal conditions, and their response to theophylline stimulation was slightly lower. ICCs generated from cryopreserved fragments were able to mature morphologically and functionally in vivo after transplantation into athymic nude mice. However, the level and magnitude of the C-peptide response did not equal that of grafted freshly cultured ICCs. Our results indicate that it is possible to generate ICCs from cryopreserved human fetal pancreas with the capacity, after transplantation, to release insulin appropriately in response to glucose. However, possibly because undifferentiated pancreatic cells may be particularly vulnerable to cryopreservation, current methods may need to be improved for optimal tissue retrieval.

Mitogen-Independent DNA Synthesis by Fetal Rat Hepatocytes in Primary Culture

We have identified conditions under which late gestation fetal rat hepatocytes in primary culture proliferate in the absence of serum, polypeptide growth factors, or insulin. Fetal hepatocytes, cultured in defined minimal essential medium (MEM) with hydrocortisone, synthesized DNA within the first 6 h after plating and for up to 72 h. Rates of thymidine incorporation into DNA by fetal hepatocytes exceeded peak rates seen with adult rat hepatocytes. The latter were quiescent following isolation, with DNA synthesis only occurring after 48 h exposure to insulin plus epidermal growth factor. Although they exhibited a high rate of DNA synthesis, the fetal hepatocytes retained sensitivity to added mitogens; DNA synthesis was stimulated three- to fourfold by subnanomolar concentrations of TGF-α. Fetal hepatocytes also were sensitive to the growth inhibitory effects of TGF-β at concentrations below 10 pM. Finally, ontogenic changes in serum- and mitogen-independent fetal hepatocyte growth were observed, with declining rates of DNA synthesis as term approached. We speculate that the ability of fetal rat hepatocytes to synthesize DNA independent of added serum or mitogens may coincide with a proliferative in vivo phenotype.

Growth promotion of transfected hepatoma cells by liver fatty acid binding protein.

Former studies have linked hepatocyte growth with liver fatty acid binding protein (L-FABP) of rat liver cytosol. In search for the roles of L-FABP in hepatocytes, we previously stably transfected rat L-FABP sense and antisense cDNAs into rat hepatoma HTC cells that do not contain L-FABP RNA or protein, thereby providing a zero-background, homologous cell model of L-FABP-expression suitable for controlled studies of its intracellular functions in hepatocyte-derived cells. The present study demonstrates the abilities of L-FABP to promote DNA synthesis and cell growth, preserve cell morphology, extend survival, and act cooperatively with unsaturated fatty acids in the transfected hepatoma cells in the absence of serum. Following removal of serum, the three control L-FABP-nonexpressing cell lines increased in cell lines increased in cell number for 24 hr and thereafter declined, whereas the three L-FABP-expressing cell lines exhibited a 39% higher rate of DNA synthesis per cell at 24 hr and grew in cell number for 48 hr. As a result, at 72 hr there were 2.5-fold (avg.) as many L-FABP-expressing cells than L-FABP-nonexpressing cells. In addition, the L-FABP-expressing cells retained their original polygonal morphology at 48 hr, when in contrast most of the control nonexpressing cells were spherical in shape with membrane blebs. In an effort to identify the agonists that collaborate with L-FABP in the growth promotion and preservation of cell morphology, various free fatty acids were examined at 48 hr for their ability to eliminate the differences in behavior of the two cell types in the serum-free medium. The unsaturated fatty acids, oleic acid (18:1 omega 9), linoleic acid (18:2 omega 6), alpha-linolenic acid (18:3 omega 3), and arachidonic acid (20:4 omega 6), at 1 microM markedly elevated the level of DNA synthesis in the more depressed control L-FABP-nonexpressing cells and moderately raised it in the less depressed L-FABP-expressing cells. In accord, the control L-FABP-nonexpressing cells needed 10(-6)-10(-5) M linoleic acid to achieve the extent of DNA synthesis attained by the expressing cells in the absence of added fatty acid. At 10 microM linoleic acid, their levels of DNA synthesis were equal. In contrast, five saturated fatty acids had no detectable effect on DNA synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Suppression of apoptosis in normal and neoplastic human B lymphocytes by CD40 ligand is independent of Bcl‐2 induction

The tendency of isolated germinal center (GC) B cells to undergo apoptosis was suppressed by recombinant cell-bound CD40 ligand (CD40L): after 2 days at 37 degrees C, > 80% of cells remained viable in the presence of CD40L as compared to < 1% in control cultures. CD40L sustained a high rate of DNA synthesis in GC cells and was more effective than monoclonal antibody to CD40 in this regard. Group I Burkitt lymphoma (BL) cell lines induced to undergo apoptosis with anti-immunoglobulin or calcium ionophore were also protected by CD40L. In BL cells, this route of rescue was not accompanied by induction of Bc1-2 protein, the expression of which has been linked to hemopoietic cell survival. Bc1-2 was induced in GC cells responding to CD40L, but its appearance was a relatively late event not reaching significant levels over controls until day 2 of culture. Thus induction of Bc1-2 appears to be secondary to the survival signal imparted by CD40L. These findings are discussed in relation to a potential role for CD40L in supporting B cell tumors in vivo and the discovery that the molecular defect in the X-linked Hyper-IgM syndrome is targeted to the CD40L gene.

Growth stimulation of primary rat hepatocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The modulation of liver growth control by the tumor promoter, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was investigated in primary hepatocytes of adult rats. Under defined conditions in serum-free cultures, the interaction of TCDD with growth-related hormones was studied. TCDD-treatment of the cultured hepatocytes for two days caused a transient stimulation of both DNA synthesis and mitotic activity. This effect was maximal at the very low nontoxic concentration of 10(-12) M TCDD, i.e., two orders of magnitude below the optimal concentrations for induction of drug metabolizing enzymes. Growth stimulation by TCDD was dependent on the presence of growth-related hormones; in primary rat hepatocytes, TCDD acted synergistically with insulin and epidermal growth factor (EGF) and antagonized the growth inhibition by dexamethasone. Under culture conditions allowing high rates of DNA synthesis, e.g., at low concentrations of dexamethasone, in the presence of EGF plus alpha 1-adrenergic agonists or rat serum, no significant effect of TCDD on cellular growth was observed. Furthermore, TCDD failed to stimulate DNA synthesis in a rat hepatoma cell line, H4IIE, which is less sensitive to growth controlling factors than normal hepatocytes. Therefore, the results suggest that the growth modulation of primary rat hepatocytes by TCDD is the most sensitive parameter of the agent thus far observed. This effect may involve both a release from the growth inhibition caused, for instance, by glucocorticoids, as well as a direct growth-stimulating effect, synergistic to the one induced by insulin.

Enhancement of peroxisomal enzymes, cytochrome P-452 and DNA synthesis in putative preneoplastic foci of rat liver treated with the peroxisome proliferator nafenopin.

The peroxisome proliferator (PP) nafenopin (NAF) enhanced tumor development in rat liver through promotion of a subtype of putative preneoplastic cell foci, characterized by weak cytoplasmic basophilia. In order to elucidate the selective growth advantage of these weakly basophilic foci (WBF) we investigated the effects of NAF on their metabolic phenotype and DNA synthesis. In WBF, as well as in other foci subpopulations and in hepatocellular carcinomas the occurrence of five NAF-inducible enzymes, i.e. of peroxisomal beta-oxidation (acyl-CoA oxidase, bifunctional protein and thiolase), catalase and cytochrome P-452 was studied by immunohistochemical methods. In untreated livers almost all foci were stained with the same intensity as the surrounding tissue. When NAF was applied, most of the liver foci showed considerably less staining than the non-focal parenchyma in which pronounced enzyme induction had occurred. However, the subpopulation of WBF showed a more heterogeneous pattern of enzyme expression varying from less to even more than in the adjacent tissue. A similarly broad range of expression of peroxisomal enzymes was found in hepatocellular carcinomas. On average, however, the tumors exhibited less staining and lower activity of peroxisomal beta-oxidation than the surrounding parenchyma. WBF always showed higher rates of DNA synthesis than other foci subtypes and unaltered liver. In approximately one-third of these foci DNA synthesis was found to be enhanced concomitantly with elevated expression of peroxisomal beta-oxidation enzymes. In conclusion, WBF may have a selective growth advantage as they 'overrespond' to the inducing effects of NAF on DNA synthesis and peroxisomal enzymes.

The Role of Hepatotrophic Stimulation in Heterotopic Hepatocyte Transplantation

AbstractIn the future, the development of methods for transplantation of large hepatocyte numbers could provide an alternative to the established liver and split liver transplantation. The use of three dimensional prevascularized polyvinyl alcohol (PVA) scaffolds have allowed hepatocyte transplantation equivalent to a whole liver mass. This study was designed to determine if a portacaval-shunt (PCS) or a 70% hepatectomy (70% HE) enhanced engraftment and proliferation of transplanted hepatocytes in heterotopic locations.Male Lewis rats served as both donors and recipients, respectively. PVA-sponges were implanted as matrices for the transplanted hepatocytes. Recipient animals were divided into four groups. All groups received transplantation of 5×107 Hepatocytes (HCTx). In addition, group A received 70% HE, group B received a PCS plus 70% HE whereas group D received PCS. Group C as a control, received only HCTx. Quantitative morphometric analysis of hepatocyte area was performed on day 0, 3, 7 and 14 after transplantation. BrdU staining was performed to study DNA-Synthesis in the graft on day 3 and 14.PCS alone and in combination with a partial hepatectomy led to significantly greater cell area one week after transplantation compared to the partial hepatectomy alone. 70% HE resulted in significantly larger cell area than the controls. At two weeks after transplantation, these significant differences persisted with the exception of Group A and C results which were not significantly different. On day 3, BrdU staining revealed a significantly higher DNA synthesis rate in Groups A and B compared to Group C. On day 14, no statistically significant differences in levels of DNA synthesis could be observed.We conclude that hepatocytes can be successfully transplanted into PVA-devices. Engraftment and proliferation can be significantly enhanced by using portacaval shunt and partial hepatectomy as hepatotrophic stimulation.

Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.

Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.

Ureteral obstruction decreases renal prepro-epidermal growth factor and Tamm-Horsfall expression

Northern and dot-blot analysis of polyadenylated RNAs of kidney cortical and outer medullary tissue was performed in male Sprague-Dawley rats at varying times up to 24 hours after bilateral ureteral obstruction (BUO), after 24 hours of unilateral obstruction (UUO) and at varying periods after release of BUO or UUO. Pre-proEGF (preproEGF) and Tamm-Horsfall (TH) mRNA declined by four hours of BUO to virtually undetectable levels at 24 hours of ureteral obstruction. Upon release of BUO or UUO, preproEGF and TH mRNA returned slowly toward normal but remained below control levels up to four days after release of ureteral obstruction. Urinary EGF excretion paralleled these changes in renal preproEGF mRNA. Although these changes are similar to those observed during nephrotoxic and ischemic renal failure, where the expression of the immediate early genes precedes the fall in preproEGF and TH expression, no such increase in the expression of these genes occurred after bilateral ureteral obstruction. These changes in preproEGF and TH expression could also be dissociated from uremia and high rates of DNA synthesis, suggesting that ureteral obstruction itself is a sufficient cause of the reduced expression. The increase in ureteral pressure and its functional and humoral effects may each play a role in reduced preproEGF and TH expression during ureteral obstruction.

Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development.

We examine clonal murine calvarial MC3T3-E1 cells to determine if they exhibit a developmental sequence similar to osteoblasts in bone tissue, namely, proliferation of undifferentiated osteoblast precursors followed by postmitotic expression of differentiated osteoblast phenotype. During the initial phase of developmental (days 1-9 of culture), MC3T3-E1 cells actively replicate, as evidenced by the high rates of DNA synthesis and progressive increase in cell number, but maintain a fusiform appearance, fail to express alkaline phosphatase, and do not accumulate mineralized extracellular collagenous matrix, consistent with immature osteoblasts. By day 9 the cultures display cuboidal morphology, attain confluence, and undergo growth arrest. Downregulation of replication is associated with expression of osteoblast functions, including production of alkaline phosphatase, processing of procollagens to collagens, and incremental deposition of a collagenous extracellular matrix. Mineralization of extracellular matrix, which begins approximately 16 days after culture, marks the final phase of osteoblast phenotypic development. Expression of alkaline phosphatase and mineralization is time but not density dependent. Type I collagen synthesis and collagen accumulation are uncoupled in the developing osteoblast. Although collagen synthesis and message expression peaks at day 3 in immature cells, extracellular matrix accumulation is minimal. Instead, matrix accumulates maximally after 7 days of culture as collagen biosynthesis is diminishing. Thus, extracellular matrix formation is a function of mature osteoblasts. Ascorbate and beta-glycerol phosphate are both essential for the expression of osteoblast phenotype as assessed by alkaline phosphatase and mineralization of extracellular matrix. Ascorbate does not stimulate type I collagen gene expression in MC3T3-E1 cells, but it is absolutely required for deposition of collagen in the extracellular matrix. Ascorbate also induces alkaline phosphatase activity in mature cells but not in immature cells. beta-glycerol phosphate displays synergistic actions with ascorbate to further stimulate collagen accumulation and alkaline phosphatase activity in postmitotic, differentiated osteoblast-like cells. Mineralization of mature cultures requires the presence of beta-glycerol phosphate. Thus, MC3T3-E1 cells display a time-dependent and sequential expression of osteoblast characteristics analogous to in vivo bone formation. The developmental sequence associated with MC3T3-E1 differentiation should provide a useful model to study the signals that mediate the switch between proliferation and differentiation in bone cells, as well as provide a renewable culture system to examine the molecular mechanism of osteoblast maturation and the formation of bone-like extracellular matrix.