Inactivation of the cystatin E/M tumor suppressor gene in cervical cancer.

We have previously localized a cervical cancer tumor suppressor gene to a 300 kb interval of 11q13. Analysis of candidate genes revealed loss of expression of cystatin E/M, a lysosomal cysteine protease inhibitor, in 6 cervical cancer cell lines and 9 of 11 primary cervical tumors. Examination of the three exons in four cervical cancer cell lines, 19 primary tumors, and 21 normal controls revealed homozygous deletion of exon 1 sequences in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L, a lysosomal protease overexpressed in cervical cancer. Introduction of these two point mutations using site directed mutagenesis resulted in reduced binding of mutated cystatin E/M to cathepsin L. Although mutations were not observed in any cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Reexpression of cystatin E/M was observed after 5'aza 2-deoxycytidiene and/or Trichostatin A treatment of cervical cancer cell lines, HeLa and SiHa, confirming promoter hypermethylation. Ectopic expression of cystatin E/M in these two cell lines resulted in growth suppression. There was also suppression of soft agar colony formation by HeLa cells expressing the cystatin E/M gene. Reexpression of cystatin E/M resulted in decreased intracellular and extracellular expression of cathepsin L. Overexpression of cathepsin L resulted in increased cell growth which was inhibited by the reintroduction of cystatin E/M. We conclude, therefore, that cystatin E/M is a cervical cancer suppressor gene and that the gene is inactivated by somatic mutations and promoter hypermethylation.

p53-dependent Chk1 phosphorylation is required for maintenance of prolonged G2 Arrest.

Targeting checkpoint kinases has been shown to have a potential chemosensitizing effect in cancer treatment. However, inhibitors of such kinases preferentially abrogate the DNA damage-induced G2 checkpoint in p53-/- as opposed to p53+/+ cells. The mechanisms by which p53 (TP53) can prevent abrogation of the G2 checkpoint are unclear. Using normal human diploid p53+/+ and p53-/- fibroblasts as model systems, we have compared the effects of three checkpoint inhibitors, caffeine, staurosporine and UCN-01, on gamma-radiation-induced G2 arrest. The G2 arrest in p53+/+ cells was abrogated by caffeine, but not by staurosporine and UCN-01, whereas the G2 arrest in p53-/- cells was sensitive to all three inhibitors. Chk2 (CHEK1) phosphorylation was maintained in the presence of all three inhibitors in both p53+/+ and p53-/- cells. Chk1 phosphorylation was maintained only in the presence of staurosporine and UCN-01 in p53+/+ cells. In the presence of caffeine Chk1 phosphorylation was inhibited regardless of p53 status. The pathway of Chk1 phosphorylation --> Cdc25A degradation --> inhibition of cyclin B1/Cdk1 activity --> G2 arrest is accordingly resistant to staurosporine and UCN-01 in p53+/+ cells. Moreover, sustained phosphorylation of Chk1 in the presence of staurosporine and UCN-01 is strongly related to phosphorylation of p53. The present study suggests the unique role of Chk1 in preventing abrogation of the G2 checkpoint in p53+/+ cells.

Localization of deletion to a 300 Kb interval of chromosome 11q13 in cervical cancer.

Previous molecular genetic studies on HeLa cell (a cervical cancer cell line) derived non-tumorigenic and tumorigenic hybrids have localized a tumor suppressor gene to the long arm of chromosome 11. Analysis of cervical cancer cell lines using chromosome 11 specific probes showed deletion and translocation of 11q13 sequences in five out of eight cell lines. Fluorescence in situ hybridization (FISH), using 11q13 specific probes, has shown interstitial deletion of 11q13 sequences in the HeLa cells. In order to determine whether 11q13 deletions occur in primary cervical tumors, we analysed 36 tumors using 20 different microsatellite and RFLP markers. Semi automated fluorescein based allelotyping was performed to identify loss of heterozygosity (LOH) in tumors. The results showed allelic loss in 17 (47%) tumors. Three different regions of loss, one near MEN1, the second near D11S913, and the third near INT2 locus were observed. The smallest region of deletion overlap at the D11S913 locus was localized to a 300 Kb distance between D11S4908 and D11S5023. Fluorescence in situ hybridization (FISH), using 11q13 specific cosmid and BAC (bacterial artificial chromosome) probes, confirmed allelic deletion in the tumors. PCR analysis further identified homozygous deletion of 11q13 sequences in a primary tumor, in HeLa cells and in two HeLa cell derived tumorigenic hybrid cell lines. The homozygous deletion in the cell lines was mapped to a 5.7 kb sequence of 11q13. We hypothesize therefore that a putative cervical cancer tumor suppressor gene exists within the 300 kb of chromosome 11q13.

Low-dose reduction in transformation frequency compared to unirradiated controls: the role of hyper-radiosensitivity to cell death.

Calculations based on plausible parameters taken from the existing experimental database, and new measurements on the cell cycle dependence of low-dose hyper-radiosensitivity (HRS) of non-tumorigenic HeLa x skin fibroblast human hybrid cells, provide the first experimental evidence that the selective killing of a transformation-sensitive G(2)/M-phase subpopulation as a consequence of low-dose HRS could account in part for the observed reduction of induced transformation frequencies at low doses to values below that observed spontaneously. However, it is clear that other mechanisms associated with classical adaptive response, such as induced DNA repair, are also likely to be involved.

Complexity of the mechanisms of initiation and maintenance of DNA damage-induced G2-phase arrest and subsequent G1-phase arrest: TP53-dependent and TP53-independent roles.

Through a detailed study of cell cycle progression, protein expression, and kinase activity in gamma-irradiated synchronized cultures of human skin fibroblasts, distinct mechanisms of initiation and maintenance of G2-phase and subsequent G1-phase arrests have been elucidated. Normal and E6-expressing fibroblasts were used to examine the role of TP53 in these processes. While G2 arrest is correlated with decreased cyclin B1/CDC2 kinase activity, the mechanisms associated with initiation and maintenance of the arrest are quite different. Initiation of the transient arrest is TP53-independent and is due to inhibitory phosphorylation of CDC2 at Tyr15. Maintenance of the G2 arrest is dependent on TP53 and is due to decreased levels of cyclin B1 mRNA and a corresponding decline in cyclin B1 protein level. After transiently arresting in G2 phase, normal cells chronically arrest in the subsequent G1 phase while E6-expressing cells continue to cycle. The initiation of this TP53-dependent G1-phase arrest occurs despite the presence of substantial levels of cyclin D1/CDK4 and cyclin E/CDK2 kinase activities, hyperphosphoryated RB, and active E2F1. CDKN1A (also known as p21(WAF1/CIP1)) levels remain elevated during this period. Furthermore, CDKN1A-dependent inhibition of PCNA activity does not appear to be the mechanism for this early G1 arrest. Thus the inhibition of entry of irradiated cells into S phase does not appear to be related to DNA-bound PCNA complexed to CDKN1A. The mechanism of chronic G1 arrest involves the down-regulation of specific proteins with a resultant loss of cyclin E/CDK2 kinase activity.

Effect of dose-rate on induction of neoplastic transformation in vitro by low doses of 232 MeV protons.

PURPOSE: To determine the effect of dose-rate on induction of neoplastic transformation in vitro by low doses of 232 MeV protons. MATERIALS AND METHODS: The experimental system used was the human hybrid cell assay. The dose-rates examined were 50 cGy/min and 20 cGy/h. The dose-rate 20 cGy/h was chosen as this is in the range of the maximum dose-rate that can be experienced in an unshielded space environment following a solar flare. At low dose-rate (LDR), doses from 0.5-100 cGy were studied. At high dose rate (HDR), the dose range was 0.5-200 cGy. RESULTS: The data indicated no significant differences between the two dose-rates at doses up to 100 cGy. CONCLUSION: For the endpoint of neoplastic transformation in vitro, high dose-rate data may be sufficient to estimate low dose-rate effects (20 cGy/h) in the dose range up to 100 cGy from 232 MeV protons. The data are of relevance to risk estimation for space travel.

Suppression of neoplastic transformation in vitro by low doses of low LET radiation.

A major concern of exposure to low doses of radiation is the risk of cancer induction. Epidemiologic data are rarely powerful enough to accurately discriminate this risk at doses <10 cGy. In order to gain insight into events at these low doses, laboratory-based studies of relevant endpoints are required. One such endpoint is radiation-induced neoplastic transformation in vitro. Such studies can provide quantitative dose-response data, as well as insights into underlying cellular and molecular mechanisms. Data are presented that indicate that low doses of low LET radiation can suppress neoplastic transformation in vitro to levels below those seen spontaneously. Mechanisms involved include both the death of a subpopulation of cells prone to spontaneous neoplastic transformation and the induction of DNA repair. The relative contributions of these mechanisms is dose-dependent. The relevance of these observations to radiation risk estimation is discussed.

Radiation-induced neoplastic transformation in vitro, hormesis and risk assessment.

Dose-response curves for various low-LET radiation sources have consistently been demonstrated to be J-shaped for the cancer-relevant endpoint of neoplastic transformation in vitro. Most of these studies have been performed where the radiation has been delivered at intermediate to high dose-rates (30-3000 mGy/min), where the threshold dose for induction of neoplastic transformation is around 100-200 mGy. Below these doses, the transformation frequency is less than that seen spontaneously, indicative of a hormetic effect. More recently, data have been obtained for low dose rates (<0.5 mGy/min) of low-LET radiation, and again hormetic effects are apparent but with threshold doses now being >1000 mGy. Similar trends have been reported in animal experiments as well as in human epidemiologic studies. Indeed, the relative risks for induction of neoplastic transformation in vitro in the dose range 1 to 1000 mGy agree well with those for incidence of radiation-induced breast cancer and leukemia in humans. These findings support the notion that the endpoint of neoplastic transformation in vitro is a plausible endpoint to not only study mechanisms involved in response to low doses of radiation, but also to provide information of potential importance to risk assessment.

ATR dependent activation of Chk2.

ATM and ATR are essential regulators of DNA damage checkpoints in mammalian cells through their respective effectors, Chk2 and Chk1. Cross regulation of the ATM-Chk2 and ATR-Chk1 pathways is very limited, although ATM and ATR show overlapping function in a partnership and time-dependent manner. In this study, we report that Chk2 is a substrate of ATR in response to ionizing and ultraviolet radiation. ATR activation induced by ionizing radiation (IR) is weak in ATM+/+ cells. However, when ATM is inhibited by caffeine, ATR activation is markedly enhanced. Total Chk2 and Chk2 Thr68 are also hyperphosphorylated in the presence of caffeine. Both ATM+/+ and ATM-/- cells display normal ATR activation in response to UV radiation-induced DNA damage, which is caffeine sensitive. In two lines of ATM-deficient, as well as in an ATM siRNA silencing cell line, ATR is activated when the cells are exposed to IR and is able to phosphorylate Chk2 in vitro. These observations suggest that ATR is one of the kinases that is likely involved in phosphorylation of Chk2 in response to IR when ATM is deficient.

Nonlinear response for neoplastic transformation following low doses of low let radiation.

There are now several independent studies that indicate that the dose-response for the endpoint of radiation-induced neoplastic transformation in vitro is non-linear for low linear energy transfer (LET) radiation. At low doses (<10 cGy) the transformation frequency drops below that seen spontaneously. Importantly, this observation has been made using fluoroscopic energy x-rays, a commonly used modality in diagnostic radiology, the practice of which is responsible for the majority of radiation exposure to the general public. Since the transformation frequency is reduced over a large dose range (0.1 to 10cGy) it is likely that multiple mechanisms are involved and that the relative contribution of these may vary with dose. These include the killing of a subpopulation of cells prone to spontaneous transformation at the lowest doses, and the induction of DNA repair at somewhat higher doses. Protective effects of low doses of low LET radiation on other cancer-relevant endpoints in vitro and in vivo have also been observed by several independent laboratories. These observations strongly suggest that the linear-nonthreshold dose-response model is unlikely to apply to the induction of cancer by low doses of low LET radiation in humans.

Correlation of in vitro chemopreventive efficacy data from the human epidermal cell assay with animal efficacy data and clinical trial plasma levels.

The human epidermal cell (HEC) assay, which uses carcinogen exposed normal skin keratinocytes to screen for cancer prevention efficacy, was used to screen possible preventive agents. The endpoints measured were inhibition of carcinogen-induced growth and induction of involucrin, an early marker of differentiation. Sixteen of twenty agents (apigenin, apomine, budesonide, N-(2-carboxyphenyl)retinamide, ellagic acid, ibuprofen, indomethacin, melatonin, (-)-2-oxo-4-thiazolidine carboxylic acid, polyphenon E, resveratrol, beta-sitosterol, sulfasalazine, vitamin E acetate, and zileuton) were positive in at least one of the two assay endpoints. Four agents (4-methoxyphenol, naringenin, palmitoylcarnitine chloride, and silymarin) were negative in the assay. Nine of the sixteen agents were positive for both endpoints. Agents that showed the greatest response included: ellagic acid > budesonide, ibuprofen > apigenin, and quinicrine dihydrochloride. Fifty-eight of sixty-five agents that have been evaluated in the HEC assay have also been evaluated in one or more rodent bioassays for cancer prevention and several are in clinical trials for cancer prevention. The assay has an overall predictive accuracy of approximately 91.4% for efficacy in rodent cancer prevention irrespective of the species used, the tissue model, or the carcinogen used. Comparison of the efficacious concentrations in vitro to plasma levels in clinical trials show that concentrations that produced efficacy in the HEC assay were achieved in clinical studies for 31 of 33 agents for which plasma levels and/or C(max) levels were available. For two agents, 9-cis-retinoic acid (RA) and dehydroepiandrosterone (DHEA), the plasma levels greatly exceeded the highest concentration (HC) found to have efficacy in vitro. Thus, the HEC assay has an excellent predictive potential for animal efficacy and is responsive at clinically achievable concentrations.

Human Rad51C deficiency destabilizes XRCC3, impairs recombination, and radiosensitizes S/G2-phase cells.

The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3) are expressed in mitotically growing cells and are thought to play mediating roles in homologous recombination, although their precise functions remain unclear. Among the five paralogs, Rad51C was found to be a central component present in two complexes, Rad51C-XRCC3 and Rad51B-Rad51C-Rad51D-XRCC2. We have shown previously that the human Rad51C protein exhibits three biochemical activities, including DNA binding, ATPase, and DNA duplex separation. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA-cross-linking agent mitomycin C and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G(2)/M phases of the cell cycle but not in G(1) phase. Together, these results provide direct cellular evidence for the function of human Rad51C in homologous recombinational repair.

The human epithelial cell cytotoxicity assay for determining tissue specific toxicity: method modifications.

The Human Epithelial Cell Cytotoxicity (HECC) Assay (Meth Cell Sci, 22: 17-24, 2000) has been modified to include three additional cell lines and to allow protocol adjustments for slow growing cell lines. This manuscript presents methods using human epithelial cells from ten different normal human tissues including: skin, mammary, prostate, renal, bronchial, lung, oral, ecto-cervix, colon, and liver. The HECC Assay can also be used to evaluate other types of drugs, personal care products, environmental chemicals, and potential toxicants. Human epithelial cells at an early passage are seeded into multi-well dishes. The cells are exposed to multiple concentrations of each test agent. A preliminary assay using an exposure of five days at 1 mM (if soluble) and four log dilutions is used to determine the highest concentration for the HECC Assay. In the HECC Assay, cultures are exposed for three to four days. Following the exposure period, endpoint measurements for inhibition of growth, mitochondrial function, and PCNA (proliferating cell nuclear antigen) expression or albumin synthesis (hepatocytes) are made. Data are analyzed to determine the concentration that inhibited an endpoint by 50 percent (TC(50)) for each agent in each target epithelial cell line or culture and the data are compared to determine the relative sensitivity of each epithelial cell line to the test agent.

Homozygous deletions within the 11q13 cervical cancer tumor-suppressor locus in radiation-induced, neoplastically transformed human hybrid cells.

Studies on nontumorigenic and tumorigenic human cell hybrids derived from the fusion of HeLa (a cervical cancer cell line) with GM00077 (a normal skin fibroblast cell line) have demonstrated "functional" tumor-suppressor activity on chromosome 11. It has been shown that several of the neoplastically transformed radiation-induced hybrid cells called GIMs (gamma ray induced mutants), isolated from the nontumorigenic CGL1 cells, have lost one copy of the fibroblast chromosome 11. We hypothesized, therefore, that the remaining copy of the gene might be mutated in the cytogenetically intact copy of fibroblast chromosome 11. Because a cervical cancer tumor suppressor locus has been localized to chromosome band 11q13, we performed deletion-mapping analysis of eight different GIMs using a total of 32 different polymorphic and microsatellite markers on the long arm (q arm) of chromosome 11. Four irradiated, nontumorigenic hybrid cell lines, called CONs, were also analyzed. Allelic deletion was ascertained by the loss of a fibroblast allele in the hybrid cell lines. The analysis confirmed the loss of a fibroblast chromosome 11 in five of the GIMs. Further, homozygous deletion (complete loss) of chromosome band 11q13 band sequences, including that of D11S913, was observed in two of the GIMs. Detailed mapping with genomic sequences localized the homozygous deletion to a 5.7-kb interval between EST AW167735 and EST F05086. Southern blot hybridization using genomic DNA probes from the D11S913 locus confirmed the existence of homozygous deletion in the two GIM cell lines. Additionally, PCR analysis showed a reduction in signal intensity for a marker mapped 31 kb centromeric of D11S913 in four other GIMs. Finally, Northern blot hybridization with the genomic probes revealed the presence of a novel >15-kb transcript in six of the GIMs. These transcripts were not observed in the nontumorigenic hybrid cell lines. Because the chromosome 11q13 band deletions in the tumorigenic hybrid cell lines overlapped with the minimal deletion in cervical cancer, the data suggest that the same gene may be involved in the development of cervical cancer and in radiation-induced carcinogenesis. We propose that a gene localized in proximity to the homozygous deletion is the candidate tumor-suppressor gene.