Human Diploid Fibroblast Cell Line Research Articles

Human Diploid Fibroblast Cell Lines - a Model System for Studying Immunodulatory Properties of Modern Immunobiological Drugs and Viruses.

The immunomodulatory properties of immunobiological drugs Glutoxim and Phosprenyl we well as vesicular stomatitis virus and inactivated tick-borne encephalitis vaccine virus were studied using human diploid fibroblast cell line from the collection of M. P. Chumakov Federal Research Center for Research and Development of Immunobiological Products. All tested preparations exhibited immunomodulatory activity in human diploid fibroblast cell line. Glutoxim in doses of 0.1 and 0.25 μg/ml stimulated production of IL-6 and IL-10 during 24-48 h of culturing, but did not stimulate production of IL-1β. Phosprenyl, on the contrary, increased production of IL-1β and the levels of IL-6 and IL-10. Vesicular stomatitis virus stimulated the production of IL-1β, IL-6, and IL-10, while inactivated tick-borne encephalitis vaccine virus stimulated the production of cytokines IL-8 and IL-18. Immunomodulatory activity of inactivated tick-borne encephalitis vaccine virus was first demonstrated in the in vitro system.

CO ameliorates endothelial senescence induced by 5-fluorouracil through SIRT1 activation

Endothelial senescence is the main risk factor that contributes to vascular dysfunction and the progression of vascular disease. Carbon monoxide (CO) plays an important role in preventing vascular dysfunction and in maintaining vascular physiology or homeostasis. The application of exogenous CO has been shown to confer protection in several models of cardiovascular injury or disease, including hypertension, atherosclerosis, balloon-catheter injury, and graft rejection. However, the mechanism by which CO prevents endothelial senescence has been largely unexplored. The aim of this study was to evaluate the effects of CO on endothelial senescence and to investigate the possible mechanisms underlying this process. We measured the levels of senescence-associated-β-galactosidase activity, senescence-associated secretory phenotype, reactive oxygen species (ROS) production, and stress granule in human umbilical vein endothelial cells and the WI-38 human diploid fibroblast cell line. We found that 5-fluorouracil (5FU)-induced ROS generation was inhibited by CO-releasing molecules (CORM)-A1 treatment, and endothelial senescence induced by 5FU was attenuated by CORM-A1 treatment. The SIRT1 inhibitor EX527 reversed the inhibitory effect of CO on the 5FU-induced endothelial senescence. Furthermore, SIRT1 deficiency abolished the stress granule formation by CO. Our results suggest that CO alleviates the endothelial senescence induced by 5FU through SIRT1 activation and may hence have therapeutic potential for the treatment of vascular diseases.

Hypoxia inducible factor-1α regulates autophagy via the p27-E2F1 signaling pathway.

Autophagy is a highly conserved process by which the cell contents are delivered to lysosomes for degradation, or are used to provide macromolecules for energy generation under conditions of nutritional starvation. It has previously been demonstrated that cancer cells in hypoxic regions, with an oxygen concentration below the normal physiological level, express hypoxia inducible factor (HIF)-1α, in order to adapt and survive. HIF-1α is important in the regulation of oxygen homeostasis and the transcription of hundreds of genes in response to conditions of hypoxia, hence maintaining energy and redox homeostasis. To determine if HIF-1α modulates autophagy and the underlying molecular mechanisms regulating this process, the human esophageal cancer EC109 and IMR90 human diploid fibroblast cell lines were exposed to normoxic or hypoxic conditions and the expression levels of various proteins subsequently examined. Small interfering RNA was used to silence p27, in order to investigate its role in the process of HIF-1α regulated autophagy. Hypoxia induced autophagy in IMR90 cells and it was revealed that immature IMR90 cells demonstrated an increased rate of autophagy compared with mature cells. HIF-1α promoted EC109 cell autophagy via positively modulating p27, whereas silencing of p27 abolished the autophagy induced by hypoxia. The present study identified the primary components of the p27-E2F1 signaling pathway by which HIF-1α regulates autophagy. A previously unidentified mechanism is here presented, via which cancer cells may generate energy, or obtain macromolecules for survival.

Impaired ATP6V0A2 expression contributes to Golgi dispersion and glycosylation changes in senescent cells.

Many genes and signaling pathways have been found to be involved in cellular senescence program. In the present study, we have identified 16 senescence-associated genes by differential proteomic analysis of the normal human diploid fibroblast cell line, TIG-1, and focused on ATP6V0A2. The aim of this study is to clarify the role of ATP6V0A2, the causal gene for ARCL2, a syndrome of abnormal glycosylation and impaired Golgi trafficking, in cellular senescence program. Here we showed that ATP6V0A2 is critical for cellular senescence; impaired expression of ATP6V0A2 disperses the Golgi structure and triggers senescence, suggesting that ATP6V0A2 mediates these processes. FITC-lectin staining and glycoblotting revealed significantly different glycosylation structures in presenescent (young) and senescent (old) TIG-1 cells; reducing ATP6V0A2 expression in young TIG-1 cells yielded structures similar to those in old TIG-1 cells. Our results suggest that senescence-associated impaired expression of ATP6V0A2 triggers changes in Golgi structure and glycosylation in old TIG-1 cells, which demonstrates a role of ATP6V0A2 in cellular senescence program.

017 Cryomicroscopic analysis of damage mechanisms during freezing and thawing of a human diploid fibroblast cell line

Human diploid cell strains are useful for commercial production of viral vaccines. For example, the MRC-5 cell line (lung fibroblast) is used to produce vaccines against chickenpox and other childhood diseases. To support the safe mass production of such vaccines, it is necessary to cryopreserve standardized batches of concentrated cell suspension, thus ensuring a consistent supply of cells for manufacturing. However, the cryopreservation process can result in deleterious intracellular ice formation (IIF), or trigger other mechanisms that also lead to cell damage. Therefore, towards improving the efficiency of MRC-5 cryopreservation techniques, the IIF phenomenon was investigated in cell suspensions using a high-speed video cryomicroscopy system. Previous high-speed imaging studies have visualized the IIF process in adherent cells, but not in suspended cells. In the present study, the effect of cooling rate on IIF in MRC-5 cells in suspension was determined. High-speed videos were captured at recording rates of 540–1200 frames/s during freezing to −60 °C at various rates of cooling (1–120 °C/min). In the suspended cells, intracellular ice formation manifested as an advancing solidification front, with minimal change in cell transparency; the median duration of such IIF events was 1 ms during rapid cooling (120 °C/min), demonstrating the importance of high-speed imaging techniques to detect intracellular freezing. The cumulative incidence of IIF increased with increasing cooling rate, from 0% at 1 °C/min to 96% at 120 °C/min. The mean temperature of IIF was −21.7 °C ( n = 75) and −33.0 °C ( n = 853) during cooling at 15 °C/min and 120 °C/min, respectively. For MRC-5 cells cooled at 120 °C/min in the presence of 5% Me 2 SO, the mean IIF temperature was depressed to −46.1 °C. Whereas the majority of cells did not darken significantly during cooling, prominent cell darkening was often observed upon warming of the frozen cells. Video recordings were acquired during warming from −60 °C to 4 °C at a warming rate of 120 °C/min. In cells with intracellular ice, the probability of darkening during warming was greater than 50%, and darkening during warming appeared to be more likely for cells that had been frozen at a higher cooling rate. In slowly cooled samples, another phenomenon was observed during rapid warming: prior to melting of extracellular ice, a transient swelling was seen in many cells. Because the magnitude of these volume excursions was often large (e.g., doubling the original cell volume), it is likely that such events are damaging, and may represent a new form of slow-cooling injury. Source of funding: Funded in part by a gift from Merck & Co., Inc. to Villanova University. Conflict of interest: None declared. jens.karlsson@villanova.edu

Timeless functions independently of the Tim-Tipin complex to promote sister chromatid cohesion in normal human fibroblasts

The Timeless-Tipin complex and Claspin are mediators of the ATR-dependent activation of Chk1 in the intra-S checkpoint response to stalled DNA replication forks. Tim-Tipin and Claspin also contribute to sister chromatid cohesion (SCC) in various organisms, likely through a replication-coupled process. Some models of the establishment of SCC posit that interactions between cohesin rings and replisomes could result in physiological replication stress requiring fork stabilization. The contributions of Timeless, Tipin, Claspin, Chk1 and ATR to SCC were investigated in genetically stable, human diploid fibroblast cell lines. Whereas Timeless, Tipin and Claspin showed similar contributions to UVC-induced activation of Chk1, siRNA-mediated knockdown of Timeless induced a 100-fold increase in sister chromatid discohesion, whereas the inductive effects of knocking down Tipin, Claspin and ATR were 4–20-fold. Knockdown of Chk1 did not significantly affect SCC. Consistent findings were obtained in two independently derived human diploid fibroblast lines and support a conclusion that SCC in human cells is strongly dependent on Timeless but independent of Chk1. Furthermore, the 10-fold difference in discohesion observed when depleting Timeless versus Tipin indicates that Timeless has a function in SCC that is independent of the Tim-Tipin complex, even though the abundance of Timeless is reduced when Tipin is targeted for depletion. A better understanding of how Timeless, Tipin and Claspin promote SCC will elucidate non-checkpoint functions of these proteins at DNA replication forks and inform models of the establishment of SCC.

Comparing the cytotoxic potential of <i>Withania somnifera</i> water and methanol extracts

The plant Withania somnifera (Linn.) (Solanacea) is a well-known herbal medicine used in many parts of the world. It has anti-inflammatory, antioxidant, and antitumor as well as neural protective properties. It seems as if the two most active withanolide components, namely withaferin A and withanolide D, found in methanol (MeOH) extracts, are responsible for the anti-inflammatory and antioxidant properties of the plant. The current research evaluated and compared the cytotoxic potential of water and methanol extracts of W. somnifera using a combined crystal violet MTT and Neutral Red assay. MRC-5 cells, a human embryonic lung-derived diploid fibroblast cell line, were the cells of choice. We found that the three lowest concentrations (0.007, 0.042, 0.250 microg/ml) of the plant material extracted in double distilled H(2)O and MeOH do not differ significantly in any of the assays. We therefore suggest that low concentrations of MeOH extracts (up to 0.250 microg/ml plant material) do not cause cell damage to the MRC-5 cells, however, higher levels should be avoided as cell viability and cell numbers are negatively influenced.

Low Concentrations of Hydrogen Peroxide Induce Altered Physiology of Human Diploid Fibroblasts

The objective of this study was to determine the physiological effects of low‐dose hydrogen peroxide (H2O2) treatments on the cellular physiology of GM00701, GM01354, GM01385, MRC‐5, and MRC‐9 human diploid fibroblast cell lines. A number of published reports have shown short‐term treatments of fibroblasts with H2O2 concentrations of 100 uM to 200 uM result in apoptosis. However, our preliminary studies show that 5.0 uM to 25.0 uM concentrations of H2O2 are capable of inducing apoptosis in the cell lines that were studied. One‐hour treatments of fibroblasts with these H2O2 concentrations were shown to result in statistically significant decreases in cell number. The relative cell numbers are measured 24 hours after treatment by Alamar Blue cytotoxicity assay. We also used flow cytometry to analyze the effects of H2O2 treatment on cell cycle progression in a time‐dependent manner 12 and 24 hours post‐treatment. We find a 1.5‐ to 3‐fold increase in sub G1 cells 24 hours after treatment when compared to untreated cells. The results are cell line specific. To confirm these findings, studies are currently underway to assess the effects of H2O2 treatment on intracellular reduced GSH concentrations and Poly (ADP‐ribose) polymerase (PARP) cleavage. We will assess the effects of sub‐lethal doses of H2O2 on the metabolism of low density lipoprotein (LDL) and oxidized low density lipoprotein (ox‐LDL) by flow cytometry. We will also assess the effects of sub‐lethal doses of H2O2 on cytokine‐induced IL‐6 and MCP‐1 secretion by ELISA. From our studies, we conclude that low levels of oxidative stress can have significant effects on the physiology of human fibroblasts.

Camptothecin Induces Nuclear Export of Prohibitin Preferentially in Transformed Cells through a CRM-1-dependent Mechanism

Prohibitin is a growth-suppressive protein that has multiple functions in the nucleus and the mitochondria. Our earlier studies had shown that prohibitin represses the activity of E2F transcription factors while enhancing p53-mediated transcription. At the same time, prohibitin has been implicated in mediating the proper folding of mitochondrial proteins. We had found that treatment of cells with camptothecin, a topoisomerase 1 inhibitor, led to the export of prohibitin and p53 from the nucleus to the mitochondria. Here we show that the camptothecin-induced export of prohibitin occurs preferentially in transformed cell lines, but not in untransformed or primary cells. Cells that did not display the translocation of prohibitin were refractive to the apoptotic effects of camptothecin. The translocation was mediated by a putative nuclear export signal at the C-terminal region of prohibitin; fusion of the nuclear export signal (NES) of prohibitin to green fluorescence protein led to its export from the nucleus. Leptomycin B could inhibit the nuclear export of prohibitin showing that it was a CRM-1-dependent event driven by Ran GTPase. Confirming this, prohibitin was found to physically interact with CRM-1, and this interaction was significantly higher in transformed cells. Delivery of a peptide corresponding to the NES of prohibitin prevented the export of prohibitin to cytoplasm and protected cells from apoptosis. These results suggest that the regulated translocation of prohibitin from the nucleus to the mitochondria facilitates its pleiotropic functions and might contribute to its anti-proliferative and tumor suppressive properties.

Cloning and characterization of cellular senescence-associated genes in human fibroblasts by suppression subtractive hybridization

Cellular senescence marks the end of the proliferative life span of normal cells in tissue culture and occurs after cells have undergone a certain number of population doublings (PDLs). It is accompanied by alterations in the pattern of gene expression. A specific human embryonic lung diploid fibroblast cell line, 2BS, has been studied as a model of senescence in our laboratory. Here, we report a set of cellular senescence-associated genes identified from suppression subtractive cDNA libraries from senescent and young 2BS cells. They include three novel genes and six previously identified genes of unknown function. The genes whose functions are known belong to various functional pathways that have been reported to change with the onset of senescence. These include three pre-mRNA splicing factors with reduced expression in senescent cells, indicating that the regulation of mRNA splicing is altered during cell senescence. In addition, the expression of the gene TOM1 (target of Myb 1), which has not previously been associated with cellular senescence, is shown to increase in senescent cells, and we demonstrate that the expression of antisense TOM1 gene in 2BS cells can delay the progress of senescence.

Human telomerase reverse transcriptase-immortalized MRC-5 and HCA2 human fibroblasts are fully permissive for human cytomegalovirus.

MRC-5 cells are a well-characterized human diploid fibroblast cell line approved for vaccine production and favoured for the routine propagation of human cytomegalovirus (HCMV). Ectopic expression of telomerase in fibroblasts is capable of overcoming replicative senescence induced by telomere shortening. Following delivery of the hTERT gene to MRC-5 cells using a retrovirus vector three clones were generated that (i) expressed functional telomerase activity, (ii) exhibited telomere extension and (iii) were sustained for >100 population doublings. Immortalized MRC-5-hTERT and also HCA2-hTERT human fibroblasts were both fully permissive for HCMV as determined by plaque assay, studies of virus growth kinetics and measurement of virus yields. Furthermore, telomerase-immortalized HCA2 cells proved capable of supporting the stable maintenance of an EBV-based episomal vector with efficient transgene expression when driven by the HCMV immediate early promoter. An indicator cell line suitable for the efficient detection of HCMV infection was also generated using an episome containing a reporter gene (lacZ) under the control of the HCMV beta-2.7 early promoter. Telomerase immortalization of human fibroblasts will thus facilitate the growth and detection of HCMV and also the generation of helper cell lines for the propagation of HCMV deletion mutants. Immortalization of fibroblasts by telomerase does not affect cell morphology or growth characteristics. The MRC-5-hTERT clones may therefore be suitable for additional applications in virology, cell biology, vaccine production and biotechnology.

Evidence for the expression of radiation-induced potentially lethal damage being a p53 -dependent process

Purpose : To test the hypothesis that the expression of potentially lethal damage (PLD) is a p 53 -dependent process. Materials and methods : Previously reported data on radiation sensitivity, DNA double-strand break rejoining, PLD expression and repair (PLDR) were analyzed for a group of 12 human tumor cell lines and three human diploid fibroblast cell lines. Seven of these cell lines had normal p53 gene expression while the other eight were functionally p53 -deficient. None of the cell lines was sensitive to radiation-induced apoptosis. Results : Cell lines with a normal p53 expression were more sensitive to radiation, but only when sensitivity was measured in plateau-phase cultures under conditions where PLDR was minimized. Mutation or functional inactivation of p53 by HPV E6 transformation led to a more radioresistant phenotype under these conditions as well as a significant reduction in PLDR. PLDR was inversely proportional to the percentage of radiationinduced DNA double-strand breaks rejoined in 1h in the p53 normal cell lines. Conclusions : These results suggest that the expression of PLD is primarily a p53 -dependent process. In the absence of functional p53 gene expression, the effects of PLD are minimized. These observations help clarify the role of p53 in tumor response to radiation therapy because they suggest that the effects of alterations in p53 are highly dependent on the microenvironment of the tumor, i.e. whether conditions allow for PLDR.

Senescent human fibroblasts have elevated Ku86 proteolytic cleavage activity

A proteolytic activity capable of cleaving the Ku86 subunit of Ku protein to two polypeptides, with molecular masses of 69 and 17 kDa in vitro, is present in a human diploid fibroblast (HDF) cell line. The activity is elevated in late-passaged and senescent cells, and the cleaved 69-kDa product seems able to form complex with Ku70 to bind DNA ends. However, further studies indicate that cleavage of Ku86 could be inhibited by including leupeptin in the extraction buffer, and no 69 kDa variant was evident in the cell. In fact, the levels of Ku86, Ku70 and DNA-end binding activity of Ku remained unchanged during replicative senescence. Thus, this study reveals an intriguing protease in HDFs, and also indicates that inconsistent results of Ku86 expression will be obtained if the protease activity is not completely inhibited.

The growth arrest and downregulation of c-myc transcription induced by ceramide are related events dependent on p21 induction, Rb underphosphorylation and E2F sequestering.

Ceramide is an intracellular lipid mediator generated through the sphingomyelin cycle in response to several extracellular signals. Ceramide has been shown to induce growth inhibition, c-myc downmodulation and apoptosis. In this paper we examined the mechanism by which ceramide induces growth suppression and the role of the G1-CDK/pRb/E2F pathway in this process. The addition of exogenous, cell-permeable C2-ceramide to the Hs 27 human diploid fibroblast cell line resulted in a dose-dependent induction of the p21WAF1/CIP1/Sdi1 kinase inhibitor with reduction of cyclin-D1 associated kinase activity. Furthermore, significant dephosphorylation of pRb was observed, with increased association of pRb and the E2F transcription factor into a transcriptionally inactive complex. Ceramide was also capable of inhibiting the transcriptional activity of a CAT reporter vector driven by E2F binding sites containing c-myc promoter transfected into Hs 27 cells. The requirement of the pRb protein for ceramide-induced c-myc downregulation was supported by the failure of ceramide to inhibit promoter activity in HeLa cells, in which pRb function is abrogated by the presence of the E7 Papilloma virus oncoprotein, and in pRb-deleted SAOS2 AT cells. Ceramide-induced downregulation of the c-myc promoter was restored in SAOS2 #1 cells in which a functional Rb gene was reintroduced. Our studies demonstrate that pRb dephosphorylation, induced by ceramide, is at least partly necessary for c-myc downregulation, and therefore the CDK-Rb-E2F pathway appears to be a target for the ceramide-induced modulation of cell cycle regulated gene transcription.

An accessory protein enhances both DNA binding and activity of DNA polymerase alpha isolated from normal, but not transformed, human fibroblasts.

DNA polymerase alpha/primase (pol alpha) isolated from fibroblasts established from a 66-year-old human donor (GM3529) exhibited decreased specific activity compared with pol alpha from either fetal-derived fibroblasts (WI38), or pSV3.neo-transformed GM3529 fibroblasts. The pol alpha specific activity decrease was correlated with a decreased proliferative capacity frequently seen in cells from aged donors. Pol alpha isolated from pSV3.neo-transformed GM3529 cells (GM3529T) exhibited a single isoform with about 10-fold higher specific activity than pol alpha from GM3529 cells. GM3529T pol alpha was immunoreactive with both anti-pol alpha and anti-SV40 large tumor antigen. Polymerases from GM3529 and GM3529T cells were treated with a pol alpha accessory protein, alpha AP, isolated from L1210 cells. Pol alpha from GM3529T cells showed no increase in activity in the presence of alpha AP, while pol alpha isolated from GM3529 cells exhibited about an 8-fold increase in activity after treatment with alpha AP. Double stranded SV40 DNA containing multiple ori sequences exhibited a greater decrease in electrophoretic mobility in the presence of GM3529T pol alpha than when treated with GM3529 pol alpha. In the presence of pol alpha from either GM35229 or GM3529T cells SV40 dsDNA exhibited a decrease in electrophoretic mobility, and in each instance addition of alpha AP resulted in an even greater decrease in DNA mobility. These data indicate that alpha AP increased pol alpha binding to SV40 dsDNA, or that alpha AP bound the DNA in addition to previously bound pol alpha. GM3529 pol alpha also bound non-specific, non-SV40, dsDNA, whereas GM3529T pol alpha with associated TAg did not bind the non-viral dsDNA unless alpha AP was added to the preparation. While not all human diploid fibroblast cell lines derived from aged human donors necessarily exhibit decreased proliferative capacity compared with cells from young donors, decreased specific activity associated with a decline in cellular DNA synthesis is typical of pol alpha from cells derived from aged human donors. We suggest that a decrease in endogenous alpha AP interaction with pol alpha may account, in part, for the loss of DNA binding affinity and specific activity of pol alpha from GM3529 cells derived from an aged donor.

Activation of methotrexate-phenylalanine by monoclonal antibody - carboxypeptidase A conjugate for the specific treatment of ovarian cancer in vitro

Monoclonal antibody 4E3 directed against a glycosylated surface protein on human ovarian teratocarcinoma cells (CRL-1572 cell line) was conjugated to bovine carboxypeptidase A (CPA) using a 3400 Da polyethylene glycol chain bearing an N-hydroxysuccinimide group at both ends. The conjugate preparation was purified by fast protein liquid chromatography on a Superose 12/30 HR column. The 4E3-CPA conjugate was recovered in the third fraction by SDS-PAGE analysis. The specific binding of the 4E3-CPA conjugate to CRL-1572 cells was confirmed by a FACS analysis and the enzymatic activity of the conjugate remained while tested with hippuryl-L-phenylalanine. In vitro cytotoxic assays on CRL-1572 cells showed that the prodrug methotrexate-phenylalanine (MTX-Phe) alone was non-toxic (ID50 > 1000 ng ml-1) but was selectively converted to MTX when the cells were pretreated with 50 micrograms ml-1 4E3-CPA conjugate, which enhanced considerably the pharmacological activity of the prodrug with an ID50 of 70 ng ml-1. The co-culture assays with CRL-1572 and MRC-5 cells (human normal lung diploid fibroblast cell lines) demonstrated the specificity of the 4E3-CPA conjugate for the CRL-1572 cells since no cytotoxicity was observed on the MRC-5 cells. When both cell lines were mixed in ratios ranging from 1:10,000 to 1:5 (CRL-1572:MRC-5), the significant increase in the ID25 was correlated with the proportion of tumoral cells present in the cell inoculum. These results suggest that MTX-Phe combined with 4E3-CPA conjugate is a promising model for a more selective and localised anti-cancer chemotherapy based on the ADEPT concept.

Detection of polypeptides associated with the histopathological differentiation of primary lung carcinoma.

Two-dimensional polyacrylamide gel electrophoresis combined with a non-enzymatic sample preparation technique is useful for analysing clinical tumour material. Using these techniques, we analysed the relationship between the histopathological findings in primary lung malignancies and the expression of a number of unidentified polypeptides that were detected in the molecular weight region 20-35 kDa. In this study 45 cases of primary lung cancer (PLC) (21 cases of adenocarcinoma, ten cases of squamous cell carcinoma, five cases of large-cell carcinoma, one case of adenosquamous cell carcinoma, five cases of small-cell carcinoma and three cases of carcinoid tumour) were examined. For reference, a human diploid fibroblast cell line (W138) and normal peripheral lymphocytes were used. Sixteen polypeptides were judged to be associated with histopathological features. These polypeptides seem to be valuable as differentiation markers. The simultaneous evaluation of these polypeptides and some other proliferation markers (e.g. PCNA, PCNA 'satellite', Numatin/protein B23 and lamin B) seems to clarify the characteristics of each case of PLC. Furthermore, it is possible to classify PLC based on the two-dimensional electrophoresis findings, and this classification of PLC is suggested to reflect the biological features of the tumour more precisely than that based only on morphology.

Expression of p53 mRNA is proliferation‐dependent in the human fibroblast cell line WI‐38

Abstract. Although alterations in the p53 tumour suppressor gene are one of the most frequent genetic lesions occurring in human cancers, the exact function and mechanism of action of normally regulated p53 in the control of cell cycle is unclear. To clanfy further the possible role of this gene in the control of cell proliferation, the cellular level of p53‐specific mRNA and its changes during density‐dependent growth, and in different proliferation states induced by serum starvation and subsequent serum‐stimulation, were followed in WI‐38 cells, a normal human diploid fibroblast cell line. Marked differences in the expression of p53 mRNA could be observed in the different proliferation states tested. The pattern of p53 expression proved to be inversely proportional to the growth‐rate of the cultures. mRNA was considerably more abundant when cells reached confluency or were arrested by serum deprivation while serum‐stimulation caused the opposite effect. These results support the hypothesis that the p53 gene plays a role in G1 control of normal cell proliferation.

Increased expression of nucleoside diphosphate kinases/nm23 in human diploid fibroblasts transformed by SV40 large T antigen or 60Co irradiation

When the expression levels of nucleoside diphosphate (NDP) kinase/nm23 were examined in four human normal diploid fibroblast cell lines in comparison with their corresponding immortalized cells transformed by SV40 large T antigen or 60Co irradiation, mRNA levels of the two isoforms (NDP kinase A/nm23-H1, NDP kinase B/nm23-H2) were increased in the immortalized cell lines. The increase was found to be associated with increased translation products. Furthermore, the cell extracts prepared from these immortalized cell lines demonstrated slightly higher enzyme activity than those from their normal counterparts. Neither the growth state nor the in vitro aging largely affected their expression in a normal cell line (TIG-3) examined. The results suggest possible involvement of NDP kinases/nm23 in acquiring an infinite growth property of these cells.

A novel member of human tissue inhibitor of metalloproteinases (TIMP) gene family is regulated during G1 progression, mitogenic stimulation, differentiation, and senescence.

We have identified in the human diploid fibroblast cell line WI-38 a novel serum-inducible gene, mitogen-inducible gene 5 (mig-5), of the delayed-early class, which represents a new member of the family of human tissue inhibitors of metalloproteinases (TIMPs). The deduced Mig-5 protein shares the highest degree of homology with chicken TIMP-3 (74% identity) and is more distantly related to human TIMP-1 and TIMP-2 (30-38% identity), indicating that mig-5 may represent the human homolog of chicken TIMP-3. In contrast to TIMP-1 and TIMP-2, mig-5 mRNA expression is not only induced in response to mitogenic stimulation but also is subject to cell cycle regulation in normally proliferating WI-38 fibroblasts and HL-60 myeloid cells, showing a clear peak around mid-G1. In agreement with this observation, differentiation of HL-60 cells to either granulocytic or macrophage-like cells leads to increased levels of mig-5 mRNA concomitant with a block in G1. In contrast, mig-5 expression is decreased in senescent human fibroblasts, suggesting that these cells may be blocked at a stage in G1 before or after the phase of maximum mig-5 expression. Since in contrast to the vast majority of other known mitogen-inducible genes, mig-5 expression is periodically up-regulated in G1, this gene should represent an invaluable tool for the analysis of cell cycle progression, terminal differentiation, and replicative senescence.