Prolonged cell cycle arrest in irradiated human diploid skin fibroblasts: the role of nutrient deprivation

Ionizing radiation has been reported to cause an irreversible cell cycle arrest in normal human diploid fibroblasts. However, colony survival assays show that even at high doses of gamma radiation, human diploid fibroblasts do not irreversibly arrest, and that a dose-dependent fraction is capable of continued cycling. In this study, we resolve the apparent discrepancy between colony survival assays and the observed radiation-induced prolonged arrest. Using flow cytometry analysis, we have confirmed that human diploid fibroblasts do exhibit a prolonged cell cycle arrest in both G(1) and G(2)/M phases of the cell cycle. However, a single replacement of fresh growth medium stimulated a fraction of the arrested population of cells to transiently re-enter the cell cycle. Daily medium changes stimulated these irradiated human diploid fibroblasts to continue cycling until they were contact-inhibited. Thus the fraction of human diploid fibroblasts which survive radiation exposure and are capable of cycling appears to permanently arrest as a result of nutrient insufficiency. Western blot analysis demonstrated a radiation-induced elevation in TP53 (formerly known as p53) protein levels within 2 h postirradiation, followed by a decrease to levels comparable to those in unirradiated controls. The TP53 and CDKN1A (formerly known as p21) protein levels were indistinguishable after 24 h and remained elevated for a 6-day period of observation in both control and irradiated cultures. Our studies indicate that human diploid fibroblasts are capable of re-entering the cell cycle after exposure to ionizing radiation and that this re-entry is dependent on a constant supply of nutrients provided by fresh medium changes. The fraction of cells capable of resuming cell cycling is consistent with the surviving fraction of cells in colony assays.     

Evidence that high telomerase activity may induce a senescent-like growth arrest in human fibroblasts

Expression of the catalytic subunit of human telomerase (hTERT), in normal human fibroblasts allows them to escape replicative senescence. However, we have observed that populations of hTERT-immortalized human fibroblasts contain 3-20% cells with a senescent morphology. To determine what causes the appearance of these senescent-like cells, we used flow cytometry to select them from the population and analyzed them for various senescence markers, telomere length, and telomerase activity. This subpopulation of cells had elevated levels of p21 and hypophosphorylated Rb, but telomere length was similar to that of the immortal cells in the culture that was sorted. Surprisingly, telomerase activity in the senescent-like cells was significantly elevated compared with immortal cells from the same population, suggesting that high telomerase activity may induce the senescent phenotype. Furthermore, transfection of normal fibroblasts with a hTERT-expressing plasmid that confers high telomerase activity led to the induction of p21, a higher percentage of SA-beta-galactosidase-positive cells, and a greater number of cells entering growth arrest compared with controls. These results suggest that excessive telomerase activity may act as a hyperproliferative signal in cells and induce a senescent phenotype in a manner similar to that seen following overexpression of oncogenic Ras, Raf, and E2F1. Thus, there must be a critical threshold of telomerase activity that permits cell proliferation.     

Origins of G1 arrest in senescent human fibroblasts

Human diploid fibroblasts have a finite proliferative lifespan in culture, at the end of which they are ararrested with G₁ phase DNA contents. Upon serum stimulation, senescent cells are deficient in carrying out a subset of early signal transduction events such as activation of protein kinase C and induction of c-fos. Later in G₁, they uniformly fail to express late G₁ genes whose products are required for DNA synthesis, implying that they are unable to pass the R point. Failure to pass the R point may occur because senescent cells are unable to phosphorylate the retinoblastoma protein, owing to the accumulation of inactive complexes of cyclin E/Cdk2 and possibly cyclin D/Cdk4. Senescent cells contain high amounts of p21, a potent cyclin-dependent kinase inhibitor whose levels are also elevated in cells arrested in G₁ following DNA damage, suggesting that both arrests might share a common mechanism. Cell aging is accompanied by a progressive shortening of chromosomal telomeres, which could be perceived by the cells as a form of DNA damage that gives rise to the signals that inactivate the cell cycle machinery.     

Poly(ADP-ribose) metabolism in ultraviolet irradiated human fibroblasts

Exposure of human fibroblasts to 5 J/m2 of UV light resulted in a rapid increase of up to 1500% in the intracellular content of poly(ADP-ribose) and a rapid depletion of its metabolic precursor, NAD. When added just prior to UV treatment, the poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, totally blocked both the increase of poly(ADP-ribose) and decrease in NAD for up to 2.5 h. Addition of 3-aminobenzamide at the time of maximal accumulation of poly(ADP-ribose) resulted in a decrease to basal levels with a half-life of approximately 6 min. The rates of accumulation of poly(ADP-ribose) and depletion of NAD were increased in the presence of either 1-beta-arabinofuranosylcytosine or hydroxyurea. Since these agents are known to cause an additional accumulation of DNA strand breaks following UV irradiation, these data provide evidence for a mechanism in which the rate of poly(ADP-ribose) synthesis following DNA damage is regulated in intact cells by the number of DNA strand breaks. Under conditions in which the synthesis of poly(ADP-ribose) was blocked, DNA repair replication induced by UV light was neither stimulated nor inhibited.     

The induction of growth arrest in fibroblasts by SV40 T antigen

DNA tumor viruses such as SV40, Ras and papillomaviruses are the most commonly used agents in immortalization of non-hematopoietic cells, but the results are quite different. Some of them even lead instead to a senescence-like state. To verify the potential of SV40 T antigen-mediated immortalization or properties and functions of it to regulate cell growth, human dermal fibroblasts were cultured and then transfected with eukaryotic expressing plasmid psv3-neo which containing SV40 T DNA. We found that expression of oncogenic SV40 T in human dermal fibroblasts resulted in growth, arrest, earlier than the occurrence of control cell senescence, although telomerase was positive and cells grew faster than control ones in early stage following transfection. These observations suggest that SV40 T antigen can activate growth arrest in human dermal fibroblasts under normal growth condition instead of always prolonging the lifespan of fibroblasts. Moreover, high rate of cell division in early stage after transfection may be associated with the expression of telomerase activity.     

Proportions of H1 histone subspecies in human fibroblasts shift during density-dependent growth arrest independent of replicative senescence

H1 histone subspecies have been reported to vary during tissue differentiation, during aging of mammalian tissues, and as a function of DNA replicative activity. Since cultured human fibroblasts have a limited replicative life span which features arrest in the G1 phase of the cell cycle, we sought to distinguish whether any changes in the proportions of the principal H1 histone subspecies (H1A, H1B, and H1o) in late-passage fibroblasts were specific for senescent loss of replicative potential, or rather ensued as a result of prolonged inhibition of cell division. We observed an identical shift in the proportions of H1 histone subspecies during prolonged density-dependent inhibition of growth in both early-passage and late-passage cells. Since under these conditions there were no passage-specific changes, replicative senescence of human fibroblasts does not appear to involve a defect in the control of H1 histone proportions.     

Survival and mutagenesis of ultraviolet irradiated simian virus 40 in foetal human fibroblasts

Survival and mutagenesis of UV-irradiated, temperature-sensitive simian virus 40 mutants (SV40) have been studied after infection of human fibroblasts. Survival of the viral progeny obtained after 6,8 or 10 days at permissive temperature decrease as a function of the UV-dose delivered to the virus. In cels which have been pretreated with 10 Jm-2 of UV 24 hours before infection, progeny survival was increased as compared to survival in control cells. The reactivation factor varies from one to ten, depending on the number of lytic cycles carried out at permissive temperature. The level of mutation frequency, as measured by the reversion from a temperature sensitive growth phenotype towards a wild type phenotype, increases with the dose of UV-irradiation given to the virus. Moreover, the mutation frequency is increased in the viral progeny produced in UV-irradiated human cells. Similar experiments carried out with SV40-transformed human fibroblasts, which constitutively express SV40 T antigen, gave comparable results. These experiments show that, as in monkey cells, a new error-prone recovery pathway can be induced by pretreating human cells with UV-light before infection.     

Coordinate control of growth arrest states in normal human diploid fibroblasts: effect of cloned SV40 tumor antigen genes

The growth arrest states of quiescence and senescence in normal human diploid fibroblasts (HDF) are related but distinct phenomena. In an effort to (1) understand the extent to which arrest in the quiescent state and arrest in the senescent state share common regulatory mechanisms, and (2) test our hypothesis that when the quiescent phenotype is altered in HDF, then the senescent phenotype is likewise altered, we have transfected HDF with cloned SV40 tumor antigen genes. Introduction of the tumor antigen genes results in a loss of the ability to enter both the quiescent arrest state and the senescent arrest state but does not immortalize the cells or create significant aneuploidy.     

Effect of growth arrest on the doubling potential of human fibroblasts in vitro: A possible influence of the donor

Summary Population doublings versus time in culture were compared in human postnatal skin fibroblasts from normal donors, a cancer patient, and from donors suffering from Cockayne syndrome, Ataxia telangiectasia, and Fanconi’s anemia (FA). Confluent cultures were maintained in a nonproliferating state for 14 to 27 d in 0.5% serum medium. The results show that the ability of cells to resume division after a resting stage can be influenced by pathologic conditions. In arrested FA cell populations an increase of the population doublings and of the calendar time were observed. It is possible that in some cell populations the resting stage favors the expression of growth potentialities related to an instability of the cells. This work was supported by contract CRL 802030 from the I.N.S.E.R.M. and by Euratom.     

Effect of growth arrest on the doubling potential of human fibroblasts in vitro: a possible influence of the donor

Population doublings versus time in culture were compared in human postnatal skin fibroblasts from normal donors, a cancer patient, and from donors suffering from Cockayne syndrome, Ataxia telangiectasia, and Fanconi's anemia (FA). Confluent cultures were maintained in a nonproliferating state for 14 to 27 d in 0.5% serum medium. The results show that the ability of cells to resume division after a resting stage can be influenced by pathologic conditions. In arrested FA cell populations an increase of the population doublings and of the calendar time were observed. It is possible that in some cell populations the resting stage favors the expression of growth potentialities related to instability of the cells.     

Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts

Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative "senescent" state. At early population doublings (PD), fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P) via reversibly cell cycle arrested (C) to irreversibly arrested senescent cells (S). In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.     

Growth factor profile of irradiated human dermal fibroblasts using a serum-free method

Radiation therapy for cancer permanently damages tissue in the line of treatment. This study sought to establish a serum-free protocol to evaluate the growth of irradiated fibroblasts and to analyze the levels of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) compared with normal fibroblasts. One irradiated cell line of human dermal fibroblasts was established from an intraoperative specimen obtained from a patient who had undergone radiation therapy for head and neck cancer. Irradiated and normal fibroblasts were then plated in UltraCULTURE (serum and growth factor free), modified Webber's medium (bFGF 50 ng/ml, insulin-like growth factor 100 ng/ml), and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (serum with undefined basal growth factors). Irradiated cells were also seeded in UltraCULTURE with 50 and 100 ng/ml of bFGF. Cell counts were performed at 0, 1, 3, 5, and 7 days, and cell supernatants were assayed for bFGF and TGF-beta. Irradiated and normal fibroblasts exhibited stronger growth in modified Webber's medium than in Dulbecco's Modified Eagle Medium with 10% fetal bovine serum. Growth of irradiated fibroblasts under bFGF modulation was similar to their growth in Webber's medium. Furthermore, irradiated fibroblasts remained viable in a serum-free and growth factor-free environment for at least 7 days; however, their growth and autocrine growth factor production was less than that of normal cells. This confirms the results of previous studies suggesting that cells from irradiated tissue undergo cellular changes. This study provides an effective model for the first-line evaluation of agents to improve wound healing, and it helps to establish standard levels of bFGF and TGF-beta production for irradiated fibroblasts.     

Expression of human growth hormone in cultured mouse fibroblasts

The new system for the transfer and expression of foreign genes based on retroviral vectors pPS-neo, conferring neomycin resistance was constructed. The BALB/c mouse cell lines producing highly active human growth hormone (more than 7 micrograms/ml into culture medium) were constructed using these vectors. An antibody column was used to purify the growth hormone from cell culture medium. Possibilities of producers to be applied for gene therapy are discussed.     

Energetic heavy ions accelerate differentiation in the descendants of irradiated normal human diploid fibroblasts

Ionizing radiation-induced genomic instability has been demonstrated in a variety of endpoints such as delayed reproductive death, chromosome instability and mutations, which occurs in the progeny of survivors many generations after the initial insult. Dependence of these effects on the linear energy transfer (LET) of the radiation is incompletely characterized; however, our previous work has shown that delayed reductions in clonogenicity can be most pronounced at LET of 108 keV/microm. To gain insight into potential cellular mechanisms involved in LET-dependent delayed loss of clonogenicity, we investigated morphological changes in colonies arising from normal human diploid fibroblasts exposed to gamma-rays or energetic carbon ions (108 keV/microm). Exposure of confluent cultures to carbon ions was 4-fold more effective at inactivating cellular clonogenic potential and produced more abortive colonies containing reduced number of cells per colony than gamma-rays. Second, colonies were assessed for clonal morphotypic heterogeneity. The yield of differentiated cells was elevated in a dose- and LET-dependent fashion in clonogenic colonies, whereas differentiated cells predominated to a comparable extent irrespective of radiation type or dose in abortive colonies. The incidence of giant or multinucleated cells was also increased but much less frequent than that of differentiated cells. Collectively, our results indicate that carbon ions facilitate differentiation more effectively than gamma-rays as a major response in the progeny of irradiated fibroblasts. Accelerated differentiation may account, at least in part, for dose- and LET-dependent delayed loss of clonogenicity in normal human diploid cells, and could be a defensive mechanism that minimizes further expansion of aberrant cells.     

Isoform-specific activation of protein kinase c in irradiated human fibroblasts and their bystander cells

Studies over the last several years have revealed the existence of a biological phenomenon known as "bystander effect", wherein cells that are not exposed to radiation elicit a similar response to that of irradiated cells. Understanding the mechanism(s) underlying the bystander effect is important not only for radiation risk assessment but also for evaluation of protocols for cancer radiotherapy. Evaluation of signaling pathways in bystander cells may provide an insight to understand the molecular mechanisms(s) responsible for this complex phenomenon. With this objective, the time course kinetics of intracellular distribution of protein kinase C (PKC isoforms PKC-betaII, PKC-alpha/beta, PKC-theta) was investigated in total and subcellular (cytosolic and nuclear) fractions of human lung fibroblast (MRC-5) cells. MRC-5 cells were either irradiated or treated with the irradiated conditioned medium collected 1h after 1 or 10 Gy of gamma-irradiation. The radiation dose selected was in the range of therapeutic usage of radiation for the human cancer treatment. Unexpectedly, bystander cells showed higher activation of protein kinase C isoforms as compared to irradiated and sham-treated control cells. Protein kinase C isoforms were more enriched in the nuclear fraction than the cytosolic fraction proteins. Induction of PKC isoforms in bystander cells are due to post-translational modifications as shown by the non-phosphorylated protein kinase C level in both irradiated and bystander cells did not differ from the sham-treated control cells. The specific activation of protein kinase C isoforms in bystander cells as demonstrated for the first time in this study may help to identify the effect of therapeutically used radiation exposure for the tumor destructions along with its implications for adjacent non-irradiated cells and organs.     

Effect of dose rate on the survival of irradiated human skin fibroblasts.

The survival of cells in density-inhibited, confluent cultures maintained at 37 degrees C was examined following exposure to 137Cs gamma rays at low dose rates (0.023 or 0.153 Gy/h) or to 60Co gamma rays at a single high dose rate (0.70-0.75 Gy/min). Cells from an ataxia telangiectasia (AT) homozygote showed no dose-rate effect, whereas a three- to fivefold increase in D0 was observed for all other cell strains exposed at low dose rates. The magnitude of the dose-rate effect did not differ significantly among cells from persons with hereditary retinoblastoma, basal cell nevus syndrome, or AT-heterozygote compared with normal cell strains, and was not related to the size of the shoulder (extrapolation number) of the survival curve. Furthermore, no differences in the capacity for the repair of potentially lethal damage during confluent holding were observed among these latter cell strains.     

Resticted growth of human cytomegalovirus in UV-irradiated WI-38 human fibroblasts.

The growth of human CMV was inhibited by uv irradiation of cells prior to infection or during the 48-hr latent period of virus replication but not after virus synthesis began. The duration of uv exposure sufficient to inhibit CMV replication was insufficient to inhibit replication of Herpes simplex and did not prevent uninfected cells from dividing normally. The effect of uv irradiation on CMV replication may have been mediated through prevention of the virus on host cell RNA(s) synthesis.     

Augmented expression of HSP72 protein in normal human fibroblasts irradiated with ultraviolet light.

Normal human fibroblasts synthesized heat shock protein (HSP) 72 constitutively and its expression was augmented 6 hours after UV irradiation. Maximum induction of HSP72 was obtained at 12 hours and HSP72 showed a punctuated distribution in nucleus. While unscheduled DNA synthesis was almost completed 12 hours after UV irradiation, the S phase fraction decreased immediately and recovered after 6 hours. Thus, HSP72 augmentation was occurred coincidentally with the recovery of S phase, and suggested that HSP72 had some function during the recovery of DNA replication inhibited after UV irradiation.