Topoisomerase IIalpha maintains genomic stability through decatenation G(2) checkpoint signaling.

Topoisomerase IIalpha (topoIIalpha) is an essential mammalian enzyme that topologically modifies DNA and is required for chromosome segregation during mitosis. Previous research suggests that inhibition of topoII decatenatory activity triggers a G(2) checkpoint response, which delays mitotic entry because of insufficient decatenation of daughter chromatids. Here we examine the effects of both topoIIalpha and topoIIbeta on decatenatory activity in cell extracts, DNA damage and decatenation G(2) checkpoint function, and the frequencies of p16(INK4A) allele loss and gain. In diploid human fibroblast lines, depletion of topoIIalpha by small-interfering RNA was associated with severely reduced decatenatory activity, delayed progression from G(2) into mitosis and insensitivity to G(2) arrest induced by the topoII catalytic inhibitor ICRF-193. Furthermore, interphase nuclei of topoIIalpha-depleted cells showed increased frequencies of losses and gains of the tumor suppressor genetic locus p16(INK4A). This study shows that the topoIIalpha protein is required for decatenation G(2) checkpoint function, and inactivation of decatenation and the decatenation G(2) checkpoint leads to abnormal chromosome segregation and genomic instability.

The human decatenation checkpoint.

Chromatid catenation is actively monitored in human cells, with progression from G(2) to mitosis being inhibited when chromatids are insufficiently decatenated. Mitotic delay was quantified in normal and checkpoint-deficient human cells during treatment with ICRF-193, a topoisomerase II catalytic inhibitor that prevents chromatid decatenation without producing topoisomerase-associated DNA strand breaks. Ataxia telangiectasia (A-T) cells, defective in DNA damage checkpoints, showed normal mitotic delay when treated with ICRF-193. The mitotic delay in response to ICRF-193 was ablated in human fibroblasts expressing an ataxia telangiectasia mutated- and rad3-related (ATR) kinase-inactive ATR allele (ATR(ki)). BRCA1-mutant HCC1937 cells also displayed a defect in ICRF-193-induced mitotic delay, which was corrected by expression of wild-type BRCA1. Phosphorylations of hCds1 or Chk1 and inhibition of Cdk1 kinase activity, which are elements of checkpoints associated with DNA damage or replication, did not occur during ICRF-193-induced mitotic delay. Over-expression of cyclin B1 containing a dominant nuclear localization signal, and inhibition of Crm1-mediated nuclear export, reversed ICRF-193-induced mitotic delay. In combination, these results imply that ATR and BRCA1 enforce the decatenation G(2) checkpoint, which may act to exclude cyclin B1/Cdk1 complexes from the nucleus. Moreover, induction of ATR(ki) produced a 10-fold increase in chromosomal aberrations, further emphasizing the vital role for ATR in genetic stability.

Aberrant cell cycle checkpoint function in transformed hepatocytes and WB-F344 hepatic epithelial stem-like cells.

Cell cycle checkpoints are barriers to carcinogenesis as they function to maintain genomic integrity. Attenuation or ablation of checkpoint function may enhance tumor formation by permitting outgrowth of unstable cells with damaged DNA. To examine the function of cell cycle checkpoints in rat hepatocarcinogenesis, we analyzed the responses of the G (1), G (2) and mitotic spindle assembly checkpoints in normal rat hepatocytes, hepatic epithelial stem-like cells (WB-F344) and transformed derivatives of both. Normal rat hepatocytes (NRH) displayed a 73% reduction in the fraction of nuclei in early S-phase 6-8 h following 8 Gy of ionizing radiation (IR) as a quantitative measure of G (1) checkpoint function. Chemically and virally transformed hepatocyte lines displayed significant attenuation of G (1) checkpoint function, ranging from partial to complete ablation. WB-F344 rat hepatic epithelial cell lines at low, mid and high passage levels expressed G (1) checkpoint function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant attenuation of G (1) checkpoint function. Attenuation of G (1) checkpoint function in transformed hepatocytes and WB-F344 cells was associated with alterations in p53, ablated/attenuated induction of p21 (Waf1) by IR, as well as aberrant function of the spindle assembly checkpoint. NRH displayed 93% inhibition of mitosis 2 h after 1 Gy IR as a quantitative measure of G (2) checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G (2) checkpoint. Moreover, the parental WB-F344 line displayed significant age-related attenuation of G (2) checkpoint function. Abnormalities in the function of cell cycle checkpoints were detected in transformed hepatocytes and WB-F344 cells at stages of hepatocarcinogenesis preceding tumorigenicity, sustaining a hypothesis that aberrant checkpoint function contributes to carcinogenesis.

Enhanced S phase delay and inhibition of replication of an undamaged shuttle vector in UVC-irradiated xeroderma pigmentosum variant.

Xeroderma pigmentosum variant (XP-V) cells are defective in bypass replication of UVC-induced thymine dimers in DNA because they lack a novel DNA polymerase (polymerase eta). In this study the effects of UVC on S phase cells were compared in fibroblasts derived from normal donors (IDH4) and XP-V patients (CTag) and immortalized by expression of the SV40 large T antigen. These transformed fibroblasts did not activate the G(1) checkpoint or inhibit replicon initiation when damaged by UVC or gamma-rays. The transformed XP-V cells (CTag) retained the increased sensitivity to UVC-induced inhibition of DNA strand growth previously observed with their diploid counterpart. Cell cycle progression analyses showed that CTag cells displayed a stronger S phase delay than transformed fibroblasts from normal individuals (IDH4) after treatment with only 2 J/m(2) UVC. Low doses of UVC also caused a lag in CTag cell proliferation. The extent of replication of an episomal DNA (pSV011), not previously exposed to radiation, was measured after the host cells were irradiated with 1-3 J/m(2) UVC. Replication of pSV011 was barely affected in irradiated IDH4 cells. Plasmid replication was inhibited by 50% in irradiated CTag cells and this inhibition could not be accounted for by increased killing of host cells by UVC. These results suggest that even in transformed cells UVC induces DNA damage responses that are reflected in transient cell cycle arrest, delay in proliferation and inhibition of episomal DNA replication. These responses are enhanced in CTag cells, presumably because of their bypass replication defect. The accumulation of replication complexes blocked at thymine dimers and extended single-stranded regions in chromosomal DNA might sequester replication factors that are needed for plasmid and chromosomal replication. Alternatively, aberrant replication structures might activate a signal transduction pathway that down-regulates DNA synthesis.

Oxidative stress and cell cycle checkpoint function.

Oxidative stress and the damage that results from it have been implicated in a wide number of disease processes including atherosclerosis, autoimmune disorders, neuronal degeneration, and cancer. Reactive oxygen species (ROS) are ubiquitous and occur naturally in all aerobic species, coming from both exogenous and endogenous sources. ROS are quite reactive and readily damage biological molecules, including DNA. While the damaging effects of ROS on DNA have been intensively studied, the effects of oxidative damage on cell cycle checkpoint function have not. Here will we review several biologically important ROS and their sources, the cell cycle, checkpoints, and current knowledge about the effects of ROS on initiating checkpoint responses.

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 p53-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 radiation-induced DNA double-strand breaks rejoined in 1 h 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.

Apoptosis provoked by the oxidative stress inducer menadione (Vitamin K(3)) is mediated by the Fas/Fas ligand system.

Menadione, or vitamin K(3) (VK(3)), a potent oxidative stress inducer, has been recently used as an effective and remarkably safe cytotoxic drug for treatment of several human tumors. VK(3) induces apoptotic cell death through a poorly understood mechanism. Here we show for the first time that VK(3)-induced apoptosis requires the Fas/FasL system. Spleen cells from both Fas- and FasL-deficient mice (C57BL/6-lpr and C57BL/6-gld, respectively) had much lower levels of VK(3) apoptosis in vitro compared to cells from control C57BL/6 mice. VK(3) cytotoxicity toward mouse splenocytes was also blocked with a Fas-Fc fusion protein. VK(3) induced apoptosis in Jurkat cells, coincident with an increase in both Fas and FasL expression. A FasL-resistant variant of these Jurkat cells was also resistant to VK(3)-induced apoptosis. Furthermore, because VK(3) effects were inhibited by glutathione, a potent antioxidant, oxidative stress was linked to the Fas/FasL system. Moreover, since the Jurkat cell lines were p53 null, the activation of Fas/FasL system after oxidative stress apparently acted through a p53-independent pathway. The therapeutic relevance of the K vitamins has been growing in recent years; our findings offer new insight for improving and expanding their applications.

The Epstein-Barr virus protein BRLF1 activates S phase entry through E2F1 induction.

The Epstein-Barr Virus (EBV) immediate-early protein BRLF1 is one of two transactivators which mediate the switch from latent to lytic replication in EBV-infected cells. DNA viruses often modulate the function of critical cell cycle proteins to maximize the efficiency of virus replication. Here we have examined the effect of BRLF1 on cell cycle progression. A replication-deficient adenovirus expressing BRLF1 (AdBRLF1) was used to infect normal human fibroblasts and various epithelial cell lines. BRLF1 expression induced S phase entry in contact-inhibited fibroblasts and in the human osteosarcoma cell line U-2 OS. AdBRLF1 infection produced a dramatic increase in the level of E2F1 but not E2F4. In contrast, the levels of Rb, p107, and p130 were decreased in AdBRLF1-infected cells. Electrophoretic mobility shift assays confirmed an increased level of free E2F1 in the AdBRLF1-infected human fibroblasts. Consistent with the previously described effect of E2F1, AdBRLF1-infected fibroblasts had increased levels of p53 and p21 and died by apoptosis. BRLF1-induced activation of E2F1 may be required for efficient EBV lytic replication, since at least one critical viral replication gene (the viral DNA polymerase) is activated by E2F (C. Liu, N. D. Sista, and J. S. Pagano, J. Virol. 70:2545-2555, 1996).

Cell cycle control, checkpoint mechanisms, and genotoxic stress.

The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle checkpoint responses that show both similarities and differences in their molecular signaling.

Telomere shortening, telomerase expression, and chromosome instability in rat hepatic epithelial stem-like cells.

Telomeres, which are specialized structures consisting of T2AG3 repeats and proteins at the ends of chromosomes, may be essential for genomic stability. To test whether telomere length maintenance preserves genomic stability in rats (Rattus rattus and Fischer 344), we assayed telomerase activity and telomere length in the rat hepatic epithelial stem-like cell line WB-F344 during aging in vitro and in tumor-derived lines. Telomerase activity in the parental WB-F344 line was repressed at low and intermediate passage levels in vitro and reexpressed at high passages. Southern blot hybridization and quantitative fluorescence in situ hybridization analyses demonstrated that telomeres were significantly eroded at intermediate passage levels, when telomerase was repressed, and at high passage levels, when telomerase was expressed. Fluorescence in situ hybridization analysis also revealed interstitial telomeric sequences in rat chromosomes. Tumor-derived WB-F344 cell lines that express telomerase had variably shortened telomeres. Cytogenetic analyses performed on WB-F344 cells at low, intermediate, and high passages demonstrated that chromosome instability was most severe in the intermediate passage cells. These data suggest that telomere shortening during aging of rat hepatic epithelial stem-like WB-F344 cells in vitro and during selection of tumorigenic lines in vivo may destabilize chromosomes. Expression of telomerase in high passage cells appeared to partially stabilize chromosomes.

DNA signals for G2 checkpoint response in diploid human fibroblasts.

DNA (deoxyribonucleic acid) signals that induce the G2 checkpoint response were examined using proliferative secondary cultures of diploid human fibroblasts. Treatments that generated DNA double-strand breaks (DSBs) directly were effective inducers of checkpoint response, generally producing >80% inhibition of mitosis (G2 delay) and the kinase activity of M-phase-promoting factor within 2 h of treatment. Effective inducers of G2 checkpoint response included gamma-irradiation and the cancer chemotherapeutic drugs, bleomycin and etoposide. Treatments that produced DNA single-strand breaks, directly or indirectly through nucleotide excision repair, were not effective inducers of G2 delay. Ineffective treatments included incubation with camptothecin, an inhibitor of topoisomerase I (topo I), and irradiation with sublethal fluences of UVC, followed by incubation with aphidicolin. Transient severe inhibition of DNA synthesis with aphidicolin did not affect mitosis substantially, suggesting that the replication arrest input to the G2 checkpoint required more than brief inhibition of DNA synthesis. In contrast, moderate camptothecin-induced inhibition of DNA synthesis was associated with a strong inhibition of mitosis that developed 4-12 h after drug treatment. This result suggested that G2 delay was not expressed until the cells that were in S-phase at the time of treatment with camptothecin proceeded into G2. DNA damage was not necessary for induction of mitotic delay. An inhibitor of topoisomerase II (topo II), ICRF-193, which inhibits chromatid decatenation in G2 cells without damaging DNA, induced a severe inhibition of mitosis and M-phase-promoting factor kinase activity. The results suggest that DNA double-strand breaks and insufficiency of chromatid decatenation effectively induce the G2 checkpoint response, but DNA single-strand breaks do not.

p53-dependent signaling sustains DNA replication and enhances clonogenic survival in 254 nm ultraviolet-irradiated human fibroblasts.

The cyclin-dependent kinase inhibitor p21(WAF1/CIP1/SDI1/CAP20) exists in normal human fibroblasts in a quaternary complex with a cyclin, a cyclin-dependent kinase, and proliferating cell nuclear antigen. A model was proposed in which, during p53-mediated suppression of cell proliferation following treatment with 254 nm UV radiation (UVC), the enhanced expression of p21 might inhibit DNA replication by virtue of its interactions with proliferating cell nuclear antigen. To test this model, we examined the mechanisms of inhibition of DNA replication in diploid human fibroblasts that express human papillomavirus type 16 E6, which inactivates p53. E6-expressing cells were defective in G1 checkpoint responses of induction of p21 and G1 arrest after ionizing radiation-induced damage to DNA. Accordingly, E6-expressing cells were resistant to inactivation of single-cell colony formation by ionizing radiation. E6 cells also displayed normal S-phase checkpoint responses of inhibition and recovery of replicon initiation following exposure to ionizing radiation and normal ability to bypass pyrimidine dimers during DNA replication soon after UVC irradiation (i.e., postreplication repair). However, DNA replication 6 h after UVC exposure was significantly inhibited in E6 cells in comparison to isogenic controls. This failure to maintain DNA replication in S-phase cells was associated with enhanced sensitivity to inactivation of single-cell colony formation by UVC. These results indicate that the p53-induced p21 pathway is not involved in the immediate S-phase responses to radiation-induced DNA damage of inhibition of replicon initiation and translesion bypass. However, our results demonstrate that p53 and, conceivably, p21 contribute to the ability of normal human fibroblasts to sustain DNA replication activity and form colonies following UVC irradiation.

Chromosomal instability is correlated with telomere erosion and inactivation of G2 checkpoint function in human fibroblasts expressing human papillomavirus type 16 E6 oncoprotein.

Cell cycle checkpoints and tumor suppressor gene functions appear to be required for the maintenance of a stable genome in proliferating cells. In this study chromosomal destabilization was monitored in relation to telomere structure, lifespan control and G2 checkpoint function. Replicative senescence was inactivated in secondary cultures of human skin fibroblasts by expressing the human papillomavirus type 16 (HPV-16) E6 oncoprotein to inactivate p53. Chromosome aberrations were enumerated during in vitro aging of isogenic control (F5neo) and HPV-16E6-expressing (F5E6) fibroblasts. We found that structural and numerical aberrations in chromosomes were significantly increased in F5E6 cells during aging in vitro and fluorescence in situ hybridization (FISH) analysis using chromosome-specific probes demonstrated the occurrence of rearrangements involving chromosome 4 and 6 in genetically unstable F5E6 cells. Flow cytometry and karyotypic analyses revealed increased polyploidy and aneuploidy in F5E6 cells only at passages > 16, although these cells displayed defective mitotic spindle checkpoint function associated with inactivation of p53 at passages 5 and 16. G2 checkpoint function was confirmed to be gradually but progressively inactivated during in vitro aging of E6-expressing cells. Aging of F5neo fibroblasts was documented during in vitro passaging by induction of a senescence-associated marker, pH 6.0 lysosomal beta-galactosidase. F5E6 cells displayed extension of in vitro lifespan and did not induce beta-galactosidase at high passage. Erosion of telomeres during in vitro aging of telomerase-negative F5neo cells was demonstrated by Southern hybridization and by quantitative FISH analysis on an individual cell level. Telomeric signals diminished continuously as F5neo cells aged in vitro being reduced by 80% near the time of replicative senescence. Telomeric signals detected by FISH also decreased continuously during aging of telomerase-negative F5E6 cells, but telomeres appeared to be stabilized at passage 34 when telomerase was expressed. Chromosomal instability in E6-expressing cells was correlated (P < 0.05) with both loss of telomeric signals and inactivation of G2 checkpoint function. The results suggest that chromosomal stability depends upon a complex interaction among the systems of telomere length maintenance and cell cycle checkpoints.

Human topoisomerase II function, tyrosine phosphorylation and cell cycle checkpoints.

Three DNA damage-responsive cell cycle checkpoints can be shown to operate in diploid human fibroblasts. One checkpoint arrests growth in G1, another inhibits replicon initiation in S phase cells, and the third delays progression from G2 into mitosis. Progression from G2 into M is controlled in part by a cyclin-dependent kinase (cyclin B/Cdk1) that is regulated by tyrosine phosphorylation. Phosphorylation of Tyr15 on Cdk1 is inhibitory for kinase activity. Activation of cyclin B/Cdk1 at the onset of mitosis is accomplished by a phosphatase, Cdc25C, that interacts with cyclin B/Cdk1 in an autocatalytic feedback loop to remove the inhibitory phosphate at Tyr15 and activate kinase activity. DNA damage triggers G2 delay by inhibiting formation of the autocatalytic feedback loop so that dephosphorylation of Tyr15 does not occur. This suppression of activation of cyclin B/Cdk1 appears to account for the failure of damaged G2 cells to progress into mitosis. Once the damage to DNA is repaired, cells resume progression into mitosis as the cycle is re-engaged. The isoflavone genistein inhibits tyrosine kinases, including one that phosphorylates Cdk1 on Tyr15. This kinase, p56/p53lyn is rapidly induced by treatments that trigger cell cycle checkpoints (ionizing radiation, cytosine arabinoside), suggesting that this kinase may actively delay the onset of mitosis by phosphorylating Tyr15 on Cdk1. Genistein also inhibits type II DNA topoisomerase to produce a form of DNA damage that triggers all of the DNA damage-responsive cell cycle checkpoints. A brief 10 min incubation with the topoisomerase poison amsacrine was sufficient to trigger the S phase checkpoint response and inhibit replicon initiation. Inhibition of replicon initiation by 1 microM amsacrine was maximal 20-30 min after drug treatment and by 120 min, the checkpoint response had decayed to allow near control rates of replicon initiation. Topoisomerase II poisons also are powerful clastogens inducing lethal and carcinogenic chromosomal aberrations. Type II topoisomerase can break DNA in a region of chromosome 11q23 that contains the ataxia telangiectasia gene (ATM). The ATM gene controls all of the DNA damage-responsive cell cycle checkpoints. Chromosomal aberrations in 11q23 are frequently seen in acute myeloid leukemia that develops as a consequence of etoposide chemotherapy. Thus, topoisomerase poisons such as genistein may trigger chromatid breakage to inactivate AT gene function, disable cell cycle control, and induce genetic instability.

Inactivation of G2 checkpoint function and chromosomal destabilization are linked in human fibroblasts expressing human papillomavirus type 16 E6.

Chromosomal stability was linked to G2 checkpoint function in human fibroblasts expressing the human papillomavirus type 16 E6 oncoprotein. Soon after expression of E6, cells displayed an undamaged, diploid karyotype and normal mitotic delay after gamma-irradiation. As the E6-expressing cells aged through their in vitro life span, G2 checkpoint function diminished progressively. After 30-70 population doublings, 60-86% of the E6 cells displayed defective G2 checkpoint response. This attenuation of G2 checkpoint function was also associated with radiation-resistant cyclin B1/CDK1 protein kinase activity. Numerical and structural abnormalities of chromosomes developed in unirradiated E6 cells with kinetics that mirrored the loss of G2 checkpoint function. A significant correlation between inactivation of the G2 checkpoint and acquisition of chromosomal abnormalities was found, suggesting that the G2 checkpoint represents a barrier to genetic instability in cells lacking G1 checkpoint function.

Expression of telomerase in normal and malignant rat hepatic epithelia.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA repeats onto the ends of chromosomes. More than 85% of human cancers express telomerase activity and a large proportion of human hepatocellular carcinomas are positive. To study the role of telomerase expression in rat hepatocarcinogenesis, telomerase activity was assayed in various rat tissues and in two types of liver epithelial cells: hepatocytes and hepatic epithelial stem-like cells. In the present study, we demonstrate that telomerase activity in rats is tissue-specific and stable with animal aging. Liver and testis were found to be telomerase positive, spleen had low or no activity, and kidney was negative. Telomerase activity did not change significantly in 18 month-old rats compared to 2 month-old rats, but was moderately (twofold) increased during liver regeneration induced by a 2/3's partial hepatectomy. Telomerase activity was detected in isolated rat hepatocytes and low passage hepatic epithelial stem-like cells (WB-F344). Telomerase activity displayed significant variations in a propagable clone of WB-F344 cells. At low passage levels after establishment in vitro (passages 4-9) non-tumorigenic WB-F344 cells expressed telomerase activity. During further in vitro passaging these cells lost expression of telomerase. Expression of telomerase in the tumor-derived lines of WB-F344 cells but not in the selectively cycled, parental lineages of these cells suggests that there may be a role for telomerase in hepatocarcinogenesis.

TGF-alpha sustains clonal expansion by promoter-dependent, chemically initiated rat hepatocytes.

A series of promoting and non-promoting barbiturates and hydantoins were examined for their ability to sustain the growth of a phenobarbital (PB)-dependent hepatocyte line in cell culture. The effective liver tumor promoters, pentobarbital, allobarbital and 5-ethyl-5-phenylhydantoin, replaced PB and supported 6/27C1 hepatocyte colony formation in vitro at 52-87% of the level induced by PB. The weak promoters secobarbital and amobarbital supported colony formation at only 11-19% of the PB control. A significant correlation was observed for in vivo and in vitro promotion activities of barbiturates and hydantoins, indicating that clonal expansion by 6/27C1 hepatocytes was promoter-dependent. Cell density also appeared to influence hepatocyte growth in vitro. Hepatocyte colonies acquired the ability to grow in the absence of PB, such that after 10 days incubation with PB, approximately 50% of colonies continued to grow in the absence of promoter. This phenomenon of clone-size-dependent hepatocyte growth suggested the operation of an autocrine growth factor pathway. Addition of the hepatocyte mitogen and autocrine growth factor, transforming growth factor-alpha (TGF-alpha), to culture medium lacking PB induced a dose-dependent increase in 6/27C1 hepatocyte colony formation. At the optimal concentration of 3 ng/ml, TGF-alpha sustained hepatocyte clonal expansion at 84% of the level induced by 2 mM PB. Individual 6/27C1 colonies that grew from single cells in the presence of TGF-alpha were tested for promoter-dependent colony formation. Either PB or TGF-alpha supported colony formation by these cells at similar levels and when combined at optimal concentrations, the response appeared to be saturated. When these factors were tested in combination at suboptimal concentrations, the two compounds were additive for supporting colony formation by the parental 6/27C1 line. The ability of TGF-alpha to replace PB and sustain hepatocyte clonal expansion was confirmed with the tumorigenic 6/15 hepatocyte line. These results suggest that TGF-alpha and PB may promote hepatocarcinogenesis by stimulating a common signal transduction pathway.

Replication fork bypass of a pyrimidine dimer blocking leading strand DNA synthesis.

We constructed a double-stranded plasmid containing a single cis, syn-cyclobutane thymine dimer (T[c,s]T) 385 base pairs from the center of the SV40 origin of replication. This circular DNA was replicated in vitro by extracts from several types of human cells. The dimer was placed on the leading strand template of the first replication fork to encounter the lesion. Two-dimensional gel electrophoresis of replication intermediates documented the transient arrest of the replication fork by the dimer. Movement of the replication fork beyond the dimer was recognized by the appearance of a single fork arc in DNA sequences located between the T[c,s]T and the half-way point around the circular template (180 degrees from the origin). Upon completion of plasmid replication, the T[c,s]T was detected by T4 endonuclease V in about one-half (46 +/- 9%) of the closed circular daughter molecules. Our results demonstrate that extracts prepared from HeLa cells and SV40-transformed human fibroblasts (SV80, IDH4), including a cell line defective in nucleotide-excision repair (XPA), were competent for leading strand DNA synthesis opposite the pyrimidine dimer and replication fork bypass. In contrast, dimer bypass was severely impaired in otherwise replication-competent extracts from two different xeroderma pigmentosum variant cell lines.

DNA-dependent protein kinase is not required for accumulation of p53 or cell cycle arrest after DNA damage.

In response to DNA damage, cells transduce a signal that leads to accumulation and activation of p53 protein, transcriptional induction of several genes, including p21, gadd45, and gadd153, and cell cycle arrest. One hypothesis is that the signal is mediated by DNA-dependent protein kinase (DNA-PK), which consists of a catalytic subunit (DNA-PKcs) and a regulatory subunit (Ku). DNA-PK has several characteristics that support this hypothesis: Ku binds to DNA damaged by nicks or double-strand breaks, DNA-PKcs is activated when Ku binds to DNA, DNA-PK will phosphorylate p53 and other cell cycle regulatory proteins in vitro, and DNA-PKcs shares homology with ATM, which is mutated in ataxia telangiectasia and involved in signaling the p53 response to ionizing radiation. The hypothesis was tested by analyzing early passage fibroblasts from severe combined immunodeficient mice, which are deficient in DNA-PK. After exposure to ionizing radiation, UV radiation, or methyl methane-sulfonate, severe combined immunodeficient and wild-type cells were indistinguishable in their response. The accumulation of p53, induction of p21, gadd45, and gadd153, and arrest of the cell cycle in G1 and G2 occurred normally. Therefore, DNA-PK is not required for the p53 response or cell cycle arrest after DNA damage.

Liver regeneration and hepatocarcinogenesis in transforming growth factor-alpha-targeted mice.

Transforming growth factor-alpha (TGF alpha), a member of the epidermal growth factor receptor ligand family, has been implicated in the regeneration and transformation of liver. Our recent development of mice that are homozygous for a disrupted TGF alpha gene allowed us to assess the requirement for this growth factor in these complex processes. We report here that although a 70% hepatectomy produced a significant increase in hepatic TGF alpha protein levels in wild-type mice, liver regeneration nevertheless proceeded normally in the absence of the growth factor. The hepatocyte labeling indices determined for homozygous targeted and wild-type mice at 36 and 48 h after hepatectomy were comparable, and the total liver DNA to body weight ratios 8 d after hepatectomy were essentially identical for the two genotypes. These results indicate that TGF alpha, is not necessary for liver regeneration. To test its requirement in liver carcinogenesis, young mice were administered single doses of diethylnitrosamine (DEN) with or without subsequent chronic treatment with the promoting agent phenobarbital (PB). Both wild-type and homozygous mutant male mice treated with DEN or DEN plus PB developed multiple preneoplastic foci or tumors by 9 mo of age with relatively high incidence. However, while five of 88 tumors in wild-type mice attained a diameter greater than 5 mm and were classified as hepatocellular carcinomas, none of 132 tumors in livers of targeted mice reached this size. Furthermore, three of these large wild-type tumors expressed significantly elevated levels of TGF alpha protein compared with normal liver. These results indicate that TGF alpha is not required for early events in chemically induced hepatocarcinogenesis but suggest that it could be important in the progression from small preneoplastic foci to large tumors.