Diploid Fibroblast Cultures Research Articles

Peculiarities of Bone Tissue Regeneration at Application of Osteoplastic Material in Experimental Model of Purulent Bone Wound

Results of study of new osteoplastic material conditionally named «Kombas» were presented. That material consisted of nondemineralized animal collagen in a form of chips impregnated by vascular endothelium growth factor. The first step of experiment included in vitro study of the material was for cytotoxicity in diploid fibroblast cultures of 4-6 passages. At the second step purulent bone wounds were modelled in 36 Chinchilla rabbits. After debridement bone defect in the study group of animals (n=18) was filled with study material, in control group (n=18) the defect was not filled. Radiologic (X-ray, CT) and morphologic examination were performed at terms 1, 2 and 3 months. For objectification of the achieved data integral indices were proposed. Index of bone defect restoration in study group was 70% higher in 1 month, 47 % - in 2 months and 24% - in 3 months, as compared to the control group. In control group the index which characterized the completion of reparative processes exceeded that index in study group by 42% in 2 months and by 54% in 3 month of observation. Study results showed that elaborated material was not cytotoxic, possessed plasticity, marked osteoinductive and osteoconductive properties, as well as an ability to substitute bone tissue defects under conditions of purulent bone cavity in animal experiment.

Peculiarities of Bone Tissue Regeneration at Application of Osteoplastic Material in Experimental Model of Purulent Bone Wound

Results of study of new osteoplastic material conditionally named «Kombas» were presented. That material consisted of nondemineralized animal collagen in a form of chips impregnated by vascular endothelium growth factor. The first step of experiment included in vitro study of the material was for cytotoxicity in diploid fibroblast cultures of 4-6 passages. At the second step purulent bone wounds were modelled in 36 Chinchilla rabbits. After debridement bone defect in the study group of animals (n=18) was filled with study material, in control group (n=18) the defect was not filled. Radiologic (X-ray, CT) and morphologic examination were performed at terms 1, 2 and 3 months. For objectification of the achieved data integral indices were proposed. Index of bone defect restoration in study group was 70% higher in 1 month, 47 % - in 2 months and 24% - in 3 months, as compared to the control group. In control group the index which characterized the completion of reparative processes exceeded that index in study group by 42% in 2 months and by 54% in 3 month of observation. Study results showed that elaborated material was not cytotoxic, possessed plasticity, marked osteoinductive and osteoconductive properties, as well as an ability to substitute bone tissue defects under conditions of purulent bone cavity in animal experiment.

A whole-genome radiation hybrid panel for goat

A 5000rad goat–hamster panel of 121 whole-genome radiation hybrids was generated and preliminarily characterized. A normal diploid fibroblast culture from a male Boer goat was fused with a recipient thymidine kinase-deficient hamster cell line. The generated 121 radiation hybrids were grown and produced an average of 8.4mg of DNA per radiation hybrid. A SINE-PCR test showed that almost all radiation hybrids retained goat DNA. The retention frequencies of the 121 hybrids were preliminarily estimated using a collection of 42 unlinked molecular markers. An optimized panel of therein 90 radiation hybrids (CHRH5000) with an average retention frequency of 34.2% was screened on the basis of interspersed repetitive DNA content and chromosome retention frequency. The development of this WG-RH panel will provide a fundamental tool for advanced goat genome mapping studies and for mammalian comparative mapping.

Evidence for the Direct Binding of Phosphorylated p53 to Sites of DNA Breaks In vivo

Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.

Introduction of Telomerase to Ataxia Teleangiectasia Cells Can Extend Shortend Telomere and Bypass Replicative Senescence.

INTRODUCTION. Ataxia teleangiectasia (AT) is an autosomal recessive disorder characterized by progressive neurological degeneration, teleangiectasia, high sensitivity to ionizing radiation, high incidence of cancer, and premature aging of skin and hair (1). Cells derived from AT patients exhibit a variety of abnormalities in culture, such as hypersensitivity to ionizing radiation, rapid shortening of telomere length, and premature replicative senescence. Normal diploid fibroblasts in culture have a finite proliferative lifespan and enter a nondividing state termed replicative senescence (2). Ectopic expression of the catalytic subunit of telomerase, hTERT, has been shown to restore telomerase activity, stabilize telomere length and bypass replicative senescence in telomerase-negative normal fibroblasts (3) as well as in the premature aging Werner syndrome (4). In order to address a role of ATM (AT mutated) protein in the regulation of telomere length and a relationship between telomere length and premature replicative senescence in AT cells, we have examined whether fibroblasts from AT patients can be immortalized by introduction of hTERT. METHODS. Primary dermal fibroblasts from normal and AT individuals were used. Amphotrophic retrovirus expressing hTERT was created with pMX-puro-hTERT in Phenix-A cells. Cells were infected with the hTERT retrovirus, and selected in culture containing 0.5 µg / ml puromycin. Telomere length was measured by terminal restriction fragment (TRF) assay, and telomerase activity was assessed by telomeric repeat amplification protocol (TRAP) using TRAPeze kit (Intergen, Purchase, NY). RESULTS. Expression of hTERT mRNA and telomerase activity in hTERT-infected AT as well as normal cells was confirmed by RT-PCR and TRAP assays, respectively. Telomere length of hTERT-infected AT cells was extended to approximately 9 kb, compared with short telomeric DNA of about 6kb in the parent AT cells at near mortal 1 (M1) phase. Whereas parental AT cells underwent replicative senescence at about 24 population doublings (PDs), hTERT-positive AT cells survived over 40 PDs, similar to hTERT-infected normal fibroblasts (Fig. 1).

Malignant transformation of human diploid fibroblasts and suppression of their anchorage independence by introduction of chromosome 13.

Isolation of cell lines that display various degrees of transformed phenotypes may be very useful to clarify multistep mechanisms of oncogenesis, but malignant transformation of human diploid fibroblasts in culture is a very rare event. We attempted to isolate variously transformed cell lines from human diploid fibroblasts (RB) of a patient with hereditary retinoblastoma. The RB cells exhibited normal karyotypes with the exception of one copy of chromosome 13, which contained a large deletion at the q14-22 region, where the RB1 gene is located. By transfection with SV40 early genes and repeated passage, we succeeded in obtaining SV40-transfected mortal, immortalized, anchorage-independent, and tumorigenic RB cell lines. DNA fingerprinting showed that these cell lines were not contaminants, but derivatives of the original RB cells. The remaining RB1 allele may be wild-type even in the malignant cell lines, because the expression and the LT-binding ability were normal. Furthermore, we did not find any homozygous loss in 16 polymorphic markers located in the 13q14-22 region in the transformed cell lines. However, introduction of a copy of a normal chromosome 13 into the anchorage-independent cell line suppressed its anchorage-independent growth ability. All these data, together with the fact that the RB cells containing the deletion progressed to a tumorigenic state spontaneously, but normal fibroblasts did not, raise the possibility that a new tumor suppressor gene, located at 13q14-22, may play a critical role in neoplastic transformation. We conclude that these RB cell lines provide an excellent system for identification of genes involved in malignant transformation of human cells. Genes Chromosomes Cancer 26:47-53, 1999.

Grafting of genetically modified human fetal fibroblasts to produce human butyrylcholinesterase in mice

Human diploid fibroblast cultures were established from fetal skin tissue. Enzymic dissociation yielded cultures of higher growth capacity of fibroblasts than those prepared by mechanical dissociation followed by spontaneous outgrowth of cells. Transfer of recombinant human butyrylcholinesterase (BChE, EC 3.1.1.8) gene into primary human fibroblasts was achieved successfully using lipofection and retrovirus-mediated transfection. The analysis of drug-resistant colonies suggested the presence of the transcripted BChE mRNA in the cytoplasm of transfected cells. The secreted BChE protein in culture medium was assayed for enzyme activity using butyrylthiocholine as substrate. The genetically modified fibroblasts were mixed with rat tail collagen and transplanted subcutaneously and intraperitoneally to mice. Immunoreactive human BChE appeared in the plasma from the transplanted mice, reaching the top level at day 13. It was not present any longer in most of the mice 20 days later.

Aberrant p21WAF1-dependent growth arrest as the possible mechanism of abnormal resistance to ultraviolet light cytotoxicity in Li-Fraumeni syndrome fibroblast strains heterozygous for TP53 mutations.

The purpose of this study is to better understand the roles of the p53 tumor suppressor protein and the product of the p53-regulated gene p21WAF1 in the response of diploid human dermal fibroblast cultures to 254 nm ultraviolet (UV) light. We report that Li-Fraumeni syndrome (LFS) fibroblast strains heterozygous for TP53 mutation at either codon 245 or 234 exhibit markedly reduced or no expression of p21WAF1 following UV irradiation, respectively. These strains also exhibit defective nucleotide excision repair and pronounced inhibition of RNA synthesis following UV exposure, both of which are molecular hallmarks of cells derived from patients with the UV-sensitive syndrome xeroderma pigmentosum. In sharp contrast to xeroderma pigmentosum cells, however, the repair-deficient LFS cells show abnormal resistance, rather than hypersensitivity, to the killing effect of UV light. We further demonstrate that exposure of normal human fibroblasts to biologically relevant fluences (< or = 15 J/m2) of UV does not induce apoptotic cell death, indicating that UV resistant phenotype displayed by LFS strains is not associated with deregulated apoptosis. In normal fibroblasts, such treatment results in a moderate ( threefold) up-regulation of p53 protein, induction of the p21WAF1 gene, and a senescence-like growth arrest. On the other hand, exposure to > or = 20 J/m2 UV results in a striking up-regulation of p53, inhibition of p21WAF1 expression, and activation of an apoptotic pathway. We conclude that: (i) p21WAF1-mediated senescence is the principal mode of cell death induced by < or = 15 J/m2 UV light in normal human fibroblasts; (ii) there is a threshold effect for p53-dependent apoptosis and that, in normal human cells, this threshold level is induced upon expsoure to 20 J/m2 UV; (iii) the p53 signaling pathway is malfunctional in the TP53 heterozygous LFS strains examined; and (iv) the enhanced resistance to UV-induced cell killing displayed by these LFS strains is a consequence of diminished growth arrest, which is presumably mediated by p21WAF1 and not abnormalities in an apoptotic pathway.

Attenuated heat shock transcriptional response in aging: molecular mechanism and implication in the biology of aging.

A characteristic feature of aging is a progressive impairment in the ability to adapt to environmental challenges. The purpose of this review is to present the experimental evidence of an attenuated heat shock transcriptional response to heat and physiological stresses in a number of aging mammalian model systems. These include the human diploid fibroblasts in culture, whole animals and animal derived cells and cell cultures, as well as peripheral blood mononuclear cells obtained from human donors. The possibility that age-dependent changes in cellular redox status, as exemplified by the increased production of reactive oxygen inter-mediates and accumulation of oxidatively-modified proteins, affects the regulation and function of the heat shock factor 1 (HSF1) and contributes to the attenuated heat shock transcriptional response in aging cells and organisms is discussed. Given the fundamentally important role of HSPs in many aspects of protein homeostasis and signal transduction, it seems likely that the inability, or compromised ability, of aging cells and organisms to produce HSPs in response to stress would contribute to the well known increase in morbidity and mortality of the aged when challenged.

Heat shock response, heat shock transcription factor and cell aging.

A characteristic feature of aging is a progressive impairment in the ability to adapt to environmental challenges. The purpose of this article is to review the evidence of an attenuated response to heat and physiological stresses in a number of mammalian aging model systems, including the human diploid fibroblasts in culture, whole animals and animal-derived cells and cell cultures, as well as peripheral blood mononuclear cells obtained from human donors. Analyses of the regulation and function of heat shock factor 1 (HSF1), a transcription factor that mediates the response to heat shock, showed that while the relative abundance of both the hsf1 transcript and the HSF1 protein did not change as a function of age, the responsiveness of HSF1 to heat-induced activation, as measured by its trimerization and ability to bind to the heat shock element consensus sequence, was inversely related to the age of the cells used. Given the fundamentally important role of heat shock proteins (HSPs) in many aspects of protein homeostasis and signal transduction it seems likely that the inability, or compromised ability, of aging cells and organisms to activate HSF1 and produce HSPs in response to stress would contribute to the well-known increase in morbidity and mortality of the aged when challenged.

Heterogeneity of bromodeoxyuridine sensitivity of cultured cells from melanoma metastases.

Continuously growing cell cultures, testing positive for tyrosine activity, were derived from two brain and three lymph-node metastases of five patients with malignant melanoma. These cell cultures were analyzed regarding their proliferation rate with continuous bromodeoxyuridine (BrdUrd) labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry. Melanoma cell cultures are more sensitive toward BrdUrd in comparison to human diploid fibroblast cultures: 50% growth inhibition at 360 +/- 130 microM BrdUrd (range: 130-520; n = 11) vs. 650 +/- 50 microM BrdUrd (n = 3) for fibroblasts. Moreover, BrdUrd sensitivity in melanoma cells is oxygen dependent: 50% growth inhibition at 200 +/- 55 microM (range: 65-400 microM) for 20% oxygen vs. 360 +/- 130 microM BrdUrd for 5% oxygen. The cell cycle kinetic mechanisms of BrdUrd-induced growth inhibition is accumulation of cells in the G2 phase. Cultures from a single metastasis showed up to a 3-fold variation in BrdUrd sensitivity. In one of the brain metastases two populations of different ploidy level (pseudotriploid vs. pseudotetraploid) and BrdUrd sensitivity could be resolved. Thus, continuous BrdUrd labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry is a powerful tool to detect heterogeneity in proliferative capacity and drug sensitivity of cell populations within one tumor biopsy.

Primary fibroblasts from human adults as target cells for ex vivo transfection and gene therapy.

Diploid fibroblast (dFb) cultures were established from a total of 106 skin and serosa biopsies of human adults. Using an optimized enzymatic dissociation procedure, 10(11) dFb/cm2 skin were obtained from patients younger than 60 years after an average time of 89 +/- 8 days, with a mean population doubling time of 3.87 +/- 1.4 days. Enzymatic dissociation of skin biopsies yielded cultures of significantly higher growth capacity of dFb than those prepared by mechanical dissociation followed by spontaneous outgrowth of cells. The plating efficiency that may be crucial for clonal selection of transfected cells was negligible when dFb were plated without feeder cells at low density, while it was enhanced to 9-24% by the addition of a feeder layer of irradiated human embryonal fibroblasts. DFb secreted various cytokines with spontaneous release of interleukin-6 (IL-6) in high quantities of up to 20 ng/10(6) cells/24 hr. In addition, one-third of the culture secreted substantial amounts of granulocyte-macrophage colony-stimulating factor (GM-CSF), while low amounts of tumor necrosis factor-alpha (TNF-alpha) were detectable in some cases after irradiation of the cells. Comparison of various transfection methods by a transient luciferase expression assay demonstrated that receptor-mediated gene transfer was approximately 10-fold more efficient than cationic lipofection of dFb, while electroporation resulted in substantially less expression of the reporter gene. We conclude that primary dFb can be obtained reproducibly from human adults and represent a suitable target cell population for receptor-mediated gene transfer and cationic lipofection.(ABSTRACT TRUNCATED AT 250 WORDS)

Global change of gene expression at late G1/S boundary may occur in human IMR‐90 diploid fibroblasts during senescence

The hallmark of cellular aging is the failure of senescent diploid cells to enter or to complete the S phase of the cell cycle. The cause for such failure may hold the key for our understanding of the molecular basis of cellular aging. We have previously shown that aging of IMR-90 human diploid fibroblasts in culture is accompanied by a five to sevenfold decrease in both thymidine kinase activity and thymidine kinase mRNA level (Chang and Chen, 1988, J. Biol. Chem., 263:11431-11435). To examine whether attenuation of gene expression at G1/S boundary is unique for thymidine kinase or it may involve most, if not all, of other G1/S genes, we compared the expressions of two classes of G1/S genes in young and in old IMR-90 cells following serum stimulation. We found that the expression of all these genes, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), ribonucleotide reductase (PNR), proliferating cell nuclear antigen (PCNA), histone H1, histone H2A + 2B, histone H3, and histone H4, was induced to high levels in young IMR-90 cells but not in old IMR-90 cells. The mRNA levels of all G1/S genes in young cells were more than tenfold higher than that in old cells 12 hr after serum stimulation. The enzymes encoded by TS and DHFR genes and dUTPase also exhibited similar age-dependent attenuation in activities. In contrast, expression of growth-related genes such as eIF-5A, c-Ha-ras, and beta-actin did not show significant differences between young and old cells after serum stimulation. Computer analysis of the promoter region of these G1/S genes revealed an Sp-1 binding site as the most common cis-element. Taken together, our results suggest that the suppression of G1/S gene expressions during senescence may be a global phenomenon and that G1/S genes may be coordinately controlled.

A growth factor-inducible gene encodes a novel nuclear protein with zinc finger structure.

Growth factors rapidly induce numerous genes that encode regulatory proteins, many of which have been identified by cDNA cloning. In this study, differential hybridization was used to screen a cDNA library constructed from human mammary carcinoma MDA-468 cells that were stimulated with transforming growth factor beta-1 (TGF-beta 1) in the presence of cycloheximide. One of the cDNA clones that was induced by TGF-beta 1 was found to have a nucleotide sequence that predicts a 9,450-Da protein with homology to regulatory DNA-binding proteins. This clone was designated metallopan-stimulin-1 (MPS-1) because it encodes a metalloprotein whose mRNA is expressed in a wide variety of actively proliferating cells and tumor tissues. MPS-1 protein contains one "zinc finger" domain of the C4 type, similar to those present in proteins of the steroid/thyroid hormone receptor superfamily and other DNA-binding proteins. The mRNA for MPS-1 was induced in MDA-468 cells by fetal calf serum, TGF-beta 1, TGF-beta 2, and cAMP analogues. The MPS-1 gene is expressed at relatively high levels in several human carcinoma cell lines, particularly those derived from ectodermal layers, and at higher levels in melanomas (ontogenically of neural origin). In contrast, the MPS-1 mRNA is expressed at low levels in normal WI-38 human lung diploid fibroblasts in culture. We hypothesize that MPS-1 protein may play a role as a potentially important mediator of cellular proliferative responses to various growth factors and other environmental signals.

A specific CCAAT-binding protein, CBP/tk, may be involved in the regulation of thymidine kinase gene expression in human IMR-90 diploid fibroblasts during senescence.

The aging of IMR-90 human diploid fibroblasts in culture is accompanied by a 5-7 fold decrease in the level of thymidine kinase (TK) mRNA and TK activity (Chang, Z. F., and Chen, K. Y. (1988) J. Biol. Chem. 263, 11431-11435). We have employed a gel mobility shift analysis to investigate the molecular basis of the age-dependent attenuation of TK gene expression. Several cis-elements including two inverted CCAAT boxes, located at base pairs (bp) -36 and -67, and GC-rich Sp1 binding sites have been identified in the TK promoter. A 28-bp (-91 to -64) fragment containing the distal inverted CCAAT element was excised from the TK promoter to examine possible differences in nuclear protein binding between young and old IMR-90 cells. A prominent DNA-protein complex was identified in serum-stimulated young cells by a gel mobility shift assay. Competition analysis indicated that the binding was highly specific. The nuclear protein responsible for the complex formation was named CBP/tk (CCAAT Binding Protein for TK gene) since methylation interference assay showed that the inverted CCAAT box was involved in binding. The appearance of the CBP/tk-28-bp complex in IMR-90 cells was (i) serum-dependent, becoming prominent 12-24 h after serum stimulation, and (ii) age-dependent, prominent only in young but not in old IMR-90 cells. Similar serum- and age-dependent complex formations were also observed using a 67-bp fragment (-63 to +4) containing the proximal CCAAT element and a TATA box. In contrast, the binding activities for the Sp1 sequence were the same in young and old cells and appeared to be serum-independent. CBP/tk binding activity in nuclear extracts was abolished by heat (60 degrees C, 5 min) or treatment with proteinase K (0.1 microgram/ml) and sodium dodecyl sulfate (0.005%), but not by Nonidet P-40 or Triton X-100. Treatment of nuclear extracts with alkaline phosphatase or lectins (concanavalin A and wheat germ agglutinin) did not affect the binding activity. Metal chelators such as 1,10-ortho-phenanthroline (0.5 mM) inhibited the CBP/tk binding activity. Cycloheximide added to the serum-stimulated cultures at an early or mid-G1 phase inhibited the CBP/tk binding activity. The half-life of the serum-induced CBP/tk binding activity was estimated to be less than 1 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased mutagen sensitivity in human cultured fibroblasts with constitutively high micronucleus levels

The induction of micronuclei (MN) by incubation with different mutagenic agents was tested in diploid fibroblast cultures obtained from 15 probands with constitutively high MN rates (15.75–77.25 MN/500 cells; average 36.27 ± 17.60 MN) and 15 probands (controls) with low MN rates (4–13.75 MN/500 cells; average 8.97 ±2.73 MN). In order to find out whether fibroblast cultures of individuals with increased spontaneous MN levels exhibit an increased sensitivity to various agents with different genotoxic mechanisms, we studied the induction of MN in these cell cultures by ultraviolet (UV) irradiation, mitomycin C (MMC), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and benzo(a)pyrene-diol-epoxid (BPDE). In addition, we tested aphidicolin (APC), a polymerase alpha inhibitor, which is a potent inducer of common fragile sites. Probands with spontaneously high MN showed an significantly increased sensitivity to UV (p ≤ 0.005), MMC (p ≤ 0.005), and BPDE (p ≤ 0.005). No significant differences were found for MNNG and APC as compared to controls.

The Limited Proliferation of Cultured Human Diploid Cells: Regulation or Senescence?

It has been widely accepted that the limited life span of human diploid fibroblasts in culture provides a valid experimental model for the study of aging at the cellular level. In spite of innumerable investigations the underlying cause of cessation of growth is not known. Many approaches are being used to test the specific hypothesis that cells at the end of their life span produce an inhibitor of the initiation of DNA synthesis which irreversibly arrests cells in G1, rather than resulting in cell death. This implies that there is positive control of cell proliferation and that the final population of noncycling cells is in a stable state. There appears to be a basic contradiction between this interpretation of published data and the long-standing view that cells actually become progressively senescent during phase III of their growth in vitro. The DNA inhibitor theory can be criticized on a number of grounds, and there is considerable evidence that fibroblasts at the end of their life span are heterogeneous and have a very complex pleiotropic phenotype. One effect of these changes would be to prevent DNA synthesis and normal progression through the cell cycle. The conclusion is that phase III cells are indeed senescent, presumably as a result of a general failure to maintain the integrity of macromolecules and other cellular components.

Altered gene expression during cellular aging.

The limited division potential of normal human diploid fibroblasts in culture represents a model system for cellular aging. Observations indicate cellular senescence is an active process. Senescent cells, although unable to divide, are actively metabolizing. Hybrids from fusion of normal and immortal human cells exhibit limited division potential, suggesting that the phenotype of cellular senescence is dominant and supporting the hypothesis that senescence is genetically programmed. Fusion of immortal human cell lines with each other has identified four complementation groups for indefinite division. This indicates that a limited number of specific genes or processes are involved in senescence. Senescent cells express highly abundant DNA synthesis inhibitory messenger RNAs and produce a surface membrane associated protein inhibitor of DNA synthesis not expressed in young cells. Senescent cell membranes were used as immunogen to generate three monoclonal antibodies reacting specifically with senescent but not young cells in several normal human cell lines. We have also found that fibronectin messenger RNA accumulates to high levels in senescent cells. The role of these changes in gene expression in senescence is being explored.

Polyamine metabolism and cell-cycle-dependent gene expression in IMR-90 human diploid fibroblasts during senescence in culture

Aging of IMR-90 human diploid fibroblasts in culture is accompanied by specific changes of polyamine metabolism including: (a) a fivefold decrease of serum-induced activity of ornithine decarboxylase (ODC 1 EC 4.1.1.17); (b) a six to tenfold increase of polyamine catabolism; and (c) a reduction of putrescine uptake. These changes apparently led to a significant reduction of putrescine accumulation in senescent cells following serum stimulation. Since the induction of ODC is a mid-G 1 event, the change of polyamine metabolism may be related to changes of expression of other cell-cycle-dependent genes during cellular aging. In addition to ODC gene, we have examined the expression of two early G 1 genes, c-erbB and c-myc, and one late G 1/S gene thymidine kinase, at mRNA levels, in both young and old IMR-90 cells. We have also compared the enzyme activities of two late G 1/S genes, thymidine kinase and thymidylate synthetase, in young and old cells following serum stimulation. We did not observe significant changes of c-erbB, c-myc, and ODC mRNA levels during cellular senescence. However, we found that serum-induced mRNA level of thymidine kinase gene in old IMR-90 cells was significantly reduced compared to that in the young cells. Results also demonstrate that aging of IMR-90 cells was accompanied by significant decrease of both thymidine kinase and thymidylate synthetase activities. In view of the recognized importance of polyamines in growth regulation, it is possible that alteration of polyamine metabolism may contribute to the impairment of expression of some key G 1/S genes and such impairment may contribute to the ultimate loss of dividing potential in senescent cells.

Development of a new type of drug by using cell technology; preparation of human interferon-beta from human diploid fibroblast cultures.

Several strains of human fibroblast were identified as good producers of human interferon-beta (HuIFN-beta), among DIP-2 cell was one of the best. We have developed an improved microcarrier culture system for both the mass culture of such cells and the large scale HuIFN-beta production. A routine pilot plant, and successively a large plant, have been accomplished for preparation, purification and preclinical or clinical trials of HuIFN-beta. The purified and lyophilized HuIFN-beta was assayed for its safety for clinical use under the regulation of the National Institute of Health of Japan. Using this HuIFN- preparation, the clinical trials on various viral diseases and malignant tumors were started from the middle of 1979. More recently, the Ministry of Health and Welfare approved this HuIFN-beta as a new drug against melanoma, glioblastoma and chronic active B type hepatitis.