Li-Fraumeni Syndrome Fibroblasts Research Articles

Mutant p53 reactivation restricts the protumorigenic consequences of wild type p53 loss of heterozygosity in Li-Fraumeni syndrome patient-derived fibroblasts

The p53 tumor suppressor, encoded by the TP53 gene, serves as a major barrier against malignant transformation. Patients with Li-Fraumeni syndrome (LFS) inherit a mutated TP53 allele from one parent and a wild-type TP53 allele from the other. Subsequently, the wild-type allele is lost and only the mutant TP53 allele remains. This process, which is termed loss of heterozygosity (LOH), results in only mutant p53 protein expression. We used primary dermal fibroblasts from LFS patients carrying the hotspot p53 gain-of-function pathogenic variant, R248Q to study the LOH process and characterize alterations in various pathways before and after LOH. We previously described the derivation of mutant p53 reactivating peptides, designated pCAPs (p53 Conformation Activating Peptides). In this study, we tested the effect of lead peptide pCAP-250 on LOH and on its associated cellular changes. We report that treatment of LFS fibroblasts with pCAP-250 prevents the accumulation of mutant p53 protein, inhibits LOH, and alleviates its cellular consequences. Furthermore, prolonged treatment with pCAP-250 significantly reduces DNA damage and restores long-term genomic stability. pCAPs may thus be contemplated as a potential preventive treatment to prevent or delay early onset cancer in carriers of mutant p53.

Abstract 2591: Characterizing the metabolic landscape of dermal fibroblasts in Li-Fraumeni Syndrome for the prediction of cancer risk and drug response

Abstract Purpose: Li-Fraumeni Syndrome (LFS) is a genetic disorder associated with a significant risk of early-onset cancer. This condition affects 1 in 5000 individuals and is largely driven by germline mutations in the TP53 tumor suppressor gene, which has a broad spectrum of functions including metabolic regulation. While LFS is highly penetrant, there is a wide degree of variability in clinical phenotype, including age of onset and tumor type. This variability suggests a role for patient-specific genetic factors, such as the type of TP53 mutation, which may define each individual's cancer risk and response to therapy. There remains a significant clinical need for better prognostication of LFS patients to predict disease outcomes and improve treatment options. An emerging body of literature is focused on the identification of non-invasive biomarkers to stratify patient populations. To this end, dermal skin fibroblasts (DSFs) have been shown to contain patient-specific disease correlates in a variety of conditions. Importantly, it has been shown that different TP53 mutations may underlie differential metabolic patterns in LFS fibroblasts; hence, we hypothesize that the metabolic signatures of DSFs from LFS patients can be utilized as prognostic biomarkers of cancer risk and response to treatment. Methods and Results: To understand the phenotypic diversity of LFS fibroblasts, our lab created a mouse xenograft model and co-cultured human LFS-derived DSFs with a sarcoma cancer cell line. LFS fibroblasts initiated earlier tumor onset in the mice compared to DSFs from healthy individuals, suggesting that these fibroblasts may secrete tumorigenic factors. Moreover, this effect was abrogated by exposure to rapamycin, an inhibitor of the mTORC1 protein kinase, suggesting that mTORC1 activity may govern the paracrine activity of these divergent fibroblast phenotypes. Next, to explore the role of mTORC1 in LFS, we used inducible expression of mutant p53 in DSFs. p53 mutants promoted mTORC1 hyperactivation, leading to increased anabolic activity, basal respiration and ATP production, suggesting that mTORC1 may alter fibroblast metabolism in a p53-dependent manner. Overall, these data provide evidence for divergent metabolic profiles of LFS skin fibroblasts which may reflect LFS phenotype variability. Ongoing work in our lab aims to further characterize the metabolic profiles of LFS fibroblasts through RNA sequencing and metabolomic profiling. Machine learning techniques will then be employed to identify molecular signatures correlating with clinical features such as age of tumor onset. Significance: This work will advance our understanding of how metabolism may underpin the clinical heterogeneity of LFS. The discovery of metabolic biomarkers will provide prognostic information with the potential to improve the early detection and treatment of cancers in LFS patients. Citation Format: Pamela Psarianos, Camilla Giovino, Sangeetha Paramathas, Nish Patel, Rajesh Gupta, Ran Kafri, David Malkin. Characterizing the metabolic landscape of dermal fibroblasts in Li-Fraumeni Syndrome for the prediction of cancer risk and drug response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2591.

Abstract B15: Investigating the contribution of mTORC1-dependent stromal signaling to cancer onset in Li-Fraumeni syndrome

Abstract In this study, we are investigating the role of stromal mTORC1 signaling in the development of malignancy in individuals with Li-Fraumeni syndrome (LFS). LFS is a cancer predisposition syndrome that is associated with germline mutations in TP53. Individuals with LFS are faced with a significant lifetime risk of developing a wide spectrum of early-onset malignancies, thus necessitating the identification of pharmacologic agents to prevent tumor onset. Inhibitors of the mTORC1 pathway, namely rapamycin and metformin, have been shown to promote lifespan extension and delay or even prevent tumor onset in LFS mouse models. These findings are unsurprising—since wild-type p53 inhibits mTORC1—yet noteworthy, since they suggest that mTORC1 signaling plays a central role in favoring tumorigenesis in LFS. mTORC1 signaling has been implicated in promoting the protumorigenic senescence-associated secretory phenotype (SASP). The SASP constitutes the release of soluble factors, such as cytokines, growth factors, and proteases from senescent cells into the microenvironment, thus promoting oncogenesis in a paracrine manner. Interestingly, an increased incidence of senescence has been reported in LFS patients, and mutant p53 has been shown to promote the secretion of a quantitatively and qualitatively more protumorigenic SASP. As such, we believe that mTORC1 signaling in LFS stromal cells favors the production and secretion of protumorigenic factors, which may induce changes in the surrounding tissue to promote development of a precancerous niche, thus favoring tumorigenesis. Our objectives are 1) to investigate the contribution of stromal cells and stromal secreted factors in favoring tumorigenesis in LFS, and 2) to investigate the role of mTORC1 signaling in favoring these altered stromal phenotypes. To address these objectives, we co-injected patient-derived LFS fibroblasts with tumor cells of a common LFS subtype (rhabdomyosarcoma [RMS]) into immunocompromised mice and observed that tumors formed from the co-injection of RMS cells with LFS fibroblasts grew to be larger than tumors formed from co-injection with non-LFS fibroblasts. To determine whether LFS fibroblasts secrete more protumorigenic cytokines than non-LFS fibroblasts, we performed protein arrays with conditioned media (CM) from fibroblast cultures and saw that LFS fibroblasts secrete markedly higher levels of protumorigenic, SASP-related cytokines. To investigate the protumorigenic effects of fibroblast secreted factors, we treated spheroids formed from tumor cells of a common LFS subtype (osteosarcoma) with CM from LFS and non-LFS fibroblast cultures. LFS fibroblast CM had a robust proproliferative effect on spheroids, while non-LFS fibroblast CM inhibited spheroid growth. Thus far, we have observed that LFS stromal constituents favor tumor cell proliferation in vitro and in vivo, and based on the current literature, we believe that mTORC1 signaling plays a role in favoring these processes. Citation Format: Camilla M. Giovino, Nish Patel, Sangeetha Paramathas, David Malkin, Ran Kafri. Investigating the contribution of mTORC1-dependent stromal signaling to cancer onset in Li-Fraumeni syndrome [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B15.

Abstract 2032: Investigating the contribution of mTORC1-dependent signaling in the microenvironment to cancer onset in Li-Fraumeni syndrome

Abstract We are investigating the role of stromal mTORC1 signaling in the development of malignancy in individuals with Li-Fraumeni Syndrome (LFS). LFS is a hereditary cancer predisposition syndrome that is associated with germline mutations in TP53. Individuals with LFS are faced with a significant lifetime risk of developing a wide spectrum of early-onset malignancies, thus necessitating the identification of pharmacologic agents to prevent tumour onset. Chemical inhibitors of the mTORC1 pathway have been shown to promote lifespan extension and delay or even prevent tumour onset in LFS mouse models. These findings are noteworthy, since they suggest that mTORC1 signaling plays a central role in favoring tumorigenesis in LFS. mTORC1 signaling has been implicated in promoting the pro-tumorigenic senescence-associated secretory phenotype (SASP). The SASP constitutes the release of soluble factors, such as cytokines, growth factors, and proteases from senescent cells into the microenvironment, thus promoting oncogenesis in a paracrine manner. Mutant p53 has also been shown to promote the secretion of a variety of pro-tumorigenic factors from various cell types. We believe that mTORC1 signaling in LFS stromal cells favors the production and secretion of pro-tumorigenic factors, which may induce changes in the surrounding tissue to promote development of a precancerous niche, thus favoring tumorigenesis. Our objectives are to investigate the contribution of stromal cells and stromal secreted factors in favoring tumorigenesis in LFS, and to investigate the role of mTORC1 signaling in favoring these altered stromal phenotypes. To determine whether patient-derived LFS fibroblasts with TP53 mutations secrete more pro-tumorigenic cytokines than non-LFS fibroblasts, we performed protein arrays with conditioned media (CM) from fibroblast cultures. We saw that LFS fibroblasts secrete markedly higher levels of pro-tumorigenic, SASP-related cytokines. Next, we treated spheroids formed from cancer cells of a common LFS subtype (osteosarcoma) with CM from LFS and non-LFS fibroblast cultures. LFS fibroblast CM had a robust pro-proliferative effect on spheroids, while non-LFS fibroblast CM inhibited spheroid growth. To further address these aims in vivo, we co-injected LFS fibroblasts with cancer cells of a common LFS subtype [rhabdomyosarcoma (RMS)] into Nude mice. We found that tumours formed from the co-injection of RMS cells with LFS fibroblasts grew to be larger than tumours formed from co-injection with non-LFS fibroblasts. Thus far, we have observed that LFS stromal constituents favor cancer cell proliferation in vitro and in vivo. We are currently investigating the role that mTORC1 signaling plays in these processes by repeating these studies following chemical and genetic perturbation of mTORC1 signaling in the LFS fibroblasts. Citation Format: Camilla Giovino, Nish Patel, Sangeetha Paramathas, Gaetano Zafarana, Ran Kafri, David Malkin. Investigating the contribution of mTORC1-dependent signaling in the microenvironment to cancer onset in Li-Fraumeni syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2032.

Abstract 4002: Epigenetic control of a checkpoint for miRNA tolerance

Abstract The interferon (IFN) pathway controls the cellular response to viral infection and double-stranded RNA. We have found that this pathway provides a novel checkpoint for enhanced expression of miRNAs. The IFN pathway is abrogated in fibroblasts from Li-Fraumeni Syndrome (LFS) patients during spontaneous cellular immortalization, a necessary step in carcinogenesis. Interferon stimulated genes (ISGs) were down-regulated by epigenetic silencing during immortalization and some of these same ISGs were activated by replicative senescence. Bisulfite sequencing of the promoter regions of IFN-regulatory transcription factors (IRF5 and IRF7) revealed that IRF7 but not IRF5 was epigenetically silenced by methylation of CpG islands in immortal LFS cells. The re-activation of ISGs by those transcription factors is sufficient to inhibit cell proliferation and induce a senescence-related phenotype in immortal LFS cells. Similar epigenetic silencing of IRF7 also results in disruption of IFN pathway in ∼30% lung cancer cells and primary tissues. Examination of innate immunity status in normal bronchial epithelial cell line Beas2B, its ras transformed derivative BVK cells and tumor-generating clones reveals increasing loss of functional IFN pathway as cells become more cancerous. Furthermore, microarray profiling of differentially expressed microRNAs (miRNAs) in LFS cell lines revealed most miRNAs were up-regulated in IFN-pathway-defective MDAH087-10 fibroblasts compared to MDAH087-N cells with relatively normal IFN signaling. Overexpression of Dicer, a critical enzyme in miRNA biogenesis, promoted cell growth and colony formation in MDAH087-10 cells. However, double-stranded miRNA produced by Dicer enhanced the expression of ISGs in MDAH087-N cells resulting in significant cell death and reduced cell growth. In addition, manipulation of the IFN pathway in immortal LFS fibroblasts through transcription factor IRF7 reversed their response to Dicer overexpression due to altered activation of the IFN pathway. Dicer-overexpressing MDAH087-N cells contained lower levels of miRNA than vector control, and conversely much higher miRNA expression was detected in Dicer transfected MDAH087-10 cells. The higher miRNA tolerance in immortal LFS fibroblasts with a disrupted IFN pathway indicates for the first time that the IFN pathway as mediated through the transcription factor IRF7 must be disrupted to permit miRNA up-regulation to occur in early carcinogenesis. This IFN pathway appears to provide a checkpoint for miRNA level tolerance and its abrogation by epigenetic silencing leads to cellular immortalization and early events in tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4002. doi:10.1158/1538-7445.AM2011-4002

CREG1 enhances p16INK4a-induced cellular senescence

Cellular senescence is an irreversible growth arrest that is activated in normal cells upon shortening of telomere and other cellular stresses. Bypassing cellular senescence is a necessary step for cells to become immortal during oncogenic transformation. During the spontaneous immortalization of Li-Fraumeni Syndrome (LFS) fibroblasts, we found that CREG1 (Cellular Repressor of E1A-stimulated Genes 1) expression was decreased during immortalization and increased in senescence. Moreover, we found that repression of CREG1 expression occurs via an epigenetic mechanism, promoter DNA methylation. Ectopic expression of CREG1 in the immortal LFS cell lines decreases cell proliferation but does not directly induce senescence. We confirmed this in osteosarcoma and fibrosarcoma cancer cell lines, cancers commonly seen in Li-Fraumeni Syndrome. In addition, we found that p16INK4a is also downregulated in immortal cells and that coexpression of CREG1 and p16INK4a, an inhibitor of CDK4/6 and Rb phosphorylation, has a greater effect than either CREG1 and p16INK4a alone to reduce cell growth, induce cell cycle arrest and cellular senescence in immortal LFS fibroblasts, osteosarcoma and fibrosarcoma cell lines. Moreover, cooperation of CREG1 and p16INK4a inhibits the expression of cyclin A and cyclin B by inhibiting promoter activity thereby decreasing mRNA and protein levels; these proteins are required for S-phase entry and G2/M transition. In conclusion, this is the first evidence to demonstrate that CREG1 enhances p16INK4a–induced senescence by transcriptional repression of cell cycle-regulated genes.

Higher miRNA Tolerance in Immortal Li-Fraumeni Fibroblasts with Abrogated Interferon Signaling Pathway

The IFN pathway is abrogated in fibroblasts from Li-Fraumeni syndrome (LFS) patients during spontaneous cellular immortalization, a necessary step in carcinogenesis. Microarray profiling of differentially expressed microRNAs (miRNA) revealed that most miRNAs were upregulated in IFN pathway-defective MDAH087-10 fibroblasts compared with MDAH087-N cells with relatively normal IFN signaling. Overexpression of Dicer, a critical enzyme in miRNA biogenesis, promoted cell growth and colony formation in MDAH087-10 cells. However, double-stranded miRNA produced by Dicer enhanced the expression of IFN-stimulated genes in MDAH087-N cells resulting in significant cell death and reduced cell growth. Furthermore, manipulation of the IFN pathway in immortal LFS fibroblasts through transcription factor IRF7 reversed their response to Dicer overexpression due to changed IFN pathway activity. Dicer overexpressing MDAH087-N cells contained lower levels of miRNA than vector control, and conversely much higher miRNA expression was detected in Dicer-transfected MDAH087-10 cells. Therefore, cells with a defective IFN pathway have a higher miRNA tolerance than cells with normal IFN pathway. This work indicates for the first time that the IFN pathway as mediated through the transcription factor IRF7 must be disrupted to permit miRNA upregulation to occur in early carcinogenesis. The IFN pathway appears to provide a checkpoint for miRNA level tolerance and its abrogation leads to cellular immortalization.

Single‐cell analysis of p16INK4a and p21WAF1 expression suggests distinct mechanisms of senescence in normal human and Li‐Fraumeni Syndrome fibroblasts

Herein we used single-cell observation methods to gain insight into the roles of p16(INK4A) and p21(WAF1) (hereafter p16 and p21) in replicative senescence and ionizing radiation-induced accelerated senescence in human [normal, ataxia telangiectasia (AT) and Li-Fraumeni syndrome (LFS)] fibroblast strains. Cultures of all strains entered a state of replicative senescence at late passages, as evident from inhibition of growth, acquisition of flattened and enlarged cell morphology, and positive staining for senescence-associated beta-galactosidase. In addition, proliferating early-passage cultures of these strains exhibited accelerated senescence in response to ionizing radiation. Immunofluorescence microscopy revealed the heterogeneous expression of p16 in normal and AT fibroblast strains, with the majority of the cells exhibiting undetectable levels of p16 irrespective of in vitro culture age. Importantly, replicative senescence as well as accelerated senescence triggered by ionizing radiation were accompanied by sustained nuclear accumulation of p21, but did not correlate with p16 expression in p53-proficient (normal and AT) fibroblasts. In p53-deficient (LFS) fibroblasts, on the other hand, replicative senescence and ionizing radiation-triggered accelerated senescence strongly correlated with expression of p16 but not of p21. Furthermore, senescence in LFS fibroblasts was associated with genomic instability encompassing polyploidy. Our findings are compatible with a model in which p16 serves as a backup regulator of senescence, triggering this response preferentially in the absence of wild-type p53 activity. The possibility that one of the tumor-suppressor functions of p16 may be associated with genomic instability, preventing the emergence of malignant progeny from polyploid giant cells, is also supported by these results.

Relationship between DNA double-strand break rejoining and cell survival after exposure to ionizing radiation in human fibroblast strains with differing ATM/p53 status: Implications for evaluation of clinical radiosensitivity

To better understand the impact of defects in the DNA damage-surveillance network on the various cell-based assays used for the prediction of patient radiosensitivity. We examined noncancerous human fibroblast strains from individuals with ataxia telangiectasia (ataxia telangiectasia mutated [ATM] deficient) or Li-Fraumeni syndrome (p53 deficient) using the neutral comet, H2AX phosphorylation, and clonogenic survival assays. Using the comet assay, we found that, compared with normal fibroblasts, cells lacking either ATM or p53 function exhibited a reduced rate of double-strand break (DSB) rejoining early (< or =4 h) after exposure to 8 Gy of gamma-radiation and also exhibited high levels of unrejoined DSBs later after irradiation. ATM-deficient and p53-deficient fibroblasts also exhibited abnormally increased levels of phosphorylated H2AX (gamma-H2AX) at later intervals after irradiation. In the clonogenic assay, ATM-deficient cells exhibited marked radiosensitivity and p53-deficient cells had varying degrees of radioresistance compared with normal fibroblasts. Regardless of whether ataxia telangiectasia and Li-Fraumeni syndrome fibroblasts are DSB-repair deficient per se, it is apparent that p53 and ATM defects greatly influence the cellular phenotype as evidenced by the neutral comet and gamma-H2AX assays. Our data suggest that the gamma-H2AX levels observed at later intervals after irradiation may represent a reliable measure of the overall DSB rejoining capabilities of human fibroblasts. However, it appears that using this parameter as a predictor of radiosensitivity without knowledge of the cells' p53 status could lead to incorrect conclusions.

Stat1 Expression Is Not Sufficient to Regulate the Interferon Signaling Pathway in Cellular Immortalization

DNA hypermethylation in gene promoters is an epigenetic mechanism regulating gene expression in cellular immortalization, an important step in carcinogenesis. Previously, we studied the genes dysregulated during immortalization using spontaneously immortalized fibroblasts from patients with Li-Fraumeni syndrome (LFS), who carry a germline mutation in the tumor suppressor gene p53. We found that multiple interferon (IFN) signaling pathway genes were regulated by epigenetic silencing. In this study we focused on a key regulator of that pathway, the signal transducer and transcription activator 1 (Stat1) gene. Although Stat1 is downregulated after cellular immortalization and upregulated in immortal MDAH041 cells after 5-aza-2'-deoxycytidine (5-aza-dC) treatment, we detected no methylation of the Stat1 promoter region in these cells before or after immortalization. To analyze the function of Stat1 in immortalization, we expressed Stat1 in immortal MDAH041 cells by stable infection, expecting to induce IFN-regulated genes or cellular senescence or both. However, the overexpression of Stat1 alone was not sufficient to repress the proliferation rate of immortal MDAH041 cells or induce senescence in immortal MDAH041 cells. We concluded that factor(s) additional to Stat1 (whether IFN dependent or not) are required for the immortalization of LFS fibroblasts.

P53-induced up-regulation of MnSOD and GPx but not catalase increases oxidative stress and apoptosis.

p53-mediated apoptosis may involve the induction of redox-controlling genes, resulting in the production of reactive oxygen species. Microarray expression analysis of doxorubicin exposed, related human lymphoblasts, p53 wild-type (WT) Tk6, and p53 mutant WTK1 identified the p53-dependent up-regulation of manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx). Consensus p53 binding sequences were identified in human MnSOD and GPx promoter regions. A 3-fold increase in the MnSOD promoter activity was observed after the induction of p53 in Li-Fraumeni syndrome (LFS) fibroblast, TR9-7, expressing p53 under the control of a tetracycline-regulated promoter. An increased protein expression of endogenous MnSOD and GPx also positively correlated with the level of p53 induction in TR9-7 cells. However, catalase (CAT) protein expression remained unaltered after p53 induction. We also examined the expression of MnSOD, GPx, and CAT in a panel of normal or LFS fibroblasts, containing either WT or mutant p53. We found increased MnSOD enzymatic activity, MnSOD mRNA expression, and MnSOD and GPx protein in LFS fibroblasts carrying a WT p53 allele when compared with homozygous mutant p53 isogenic cells. The CAT protein level was unchanged in these cells. We observed both the release of cytochrome C and Ca(2+) from the mitochondria into the cytoplasm and an increased frequency of apoptotic cells after p53 induction in the TR9-7 cells that coincided with an increased expression of MnSOD and GPx, and the level of reactive oxygen species. The increase in apoptosis was reduced by the antioxidant N-acetylcysteine. These results identify a novel mechanism of p53-dependent apoptosis in which p53-mediated up-regulation of MnSOD and GPx, but not CAT, produces an imbalance in antioxidant enzymes and oxidative stress.

Decreased DNA Repair Efficiency by Loss or Disruption of p53 Function Preferentially Affects Removal of Cyclobutane Pyrimidine Dimers from Non-transcribed Strand and Slow Repair Sites in Transcribed Strand

The tumor suppressor protein p53 plays a central role in modulating the cellular responses to DNA damage. Several recent studies, undertaken with the whole genomic DNA or full-length gene segments, have shown that p53 is involved in nucleotide excision repair and it selectively influences the adduct removal from the non-transcribed strand in the genome. In this study, we have analyzed the damage induction at nucleotide resolution by ligase-mediated polymerase chain reaction and compared the repair of ultraviolet radiation-induced cyclobutane pyrimidine dimers within exon 8 of p53 gene in normal and Li-Fraumeni syndrome fibroblasts as well as in normal and human papillomavirus 16 E6 and E7 protein-expressing human mammary epithelial cells. The results demonstrate that (i) loss or disruption of p53 function decreases efficiency of DNA repair, by preferentially affecting the repair of non-transcribed strand and of intrinsically slow repair sites in transcribed strand; (ii) mutant p53 protein affects DNA repair, at least of non-transcribed strand, in a dominant negative manner; and (iii) pRb does not have an effect on the repair of DNA damage within transcribed or non-transcribed strand. The overall data suggest that p53 could regulate excision repair or related events through direct protein-protein interaction.

Role for p53 in the Recovery of Transcription and Protection Against Apoptosis Induced by Ultraviolet Light

We have previously suggested that the inhibition of RNA polymerase II-mediated transcription after exposure to UV light promotes the accumulation of p53 and the induction of apoptosis (Oncogene 13, 823–831). However, it was not clear whether p53 induction was contributing to apoptosis. Here we report that apoptosis is triggered at lower UV doses in p53-deficient Li-Fraumeni syndrome (LFS) and human papillomavirus (HPV) E6 expressing fibroblasts than in normal cells, suggesting that p53 can be protective against UVinduced apoptosis. There is no significant difference in the effect of UV-irradiation on the cell cycle distribution of normal and primary LFS fibroblasts. Importantly, the recovery of nascent mRNA synthesis in all p53-deficient fibroblasts is significantly impaired compared with control cells after exposure to relevant doses of UV light. Taken together, our results suggest that wild-type p53 can protect cells against UV-induced apoptosis by facilitating the recovery of transcription. Furthermore, we suggest that the capacity of cells to recover transcription after genotoxic damage is an important determinant of sensitivity to apoptosis.

Telomerase activity during spontaneous immortalization of Li-Fraumeni syndrome skin fibroblasts.

Li-Fraumeni Syndrome (LFS) is characterized by heterozygous germline mutations in the p53 gene. Accompanied by genomic instability and loss or mutation of the remaining wild type p53 allele, a low frequency of spontaneous immortalization in LFS fibroblasts occurs. It is believed that the loss of p53 wild type function contributes to immortalization of these LFS fibroblasts, but it is not clear if this is sufficient. Because stabilization of telomere length is also thought to be a necessary step in immortalization, telomerase activity, expression of the telomerase RNA component (hTR) and telomere length were anlaysed at various passages during the spontaneous immortalization of LFS skin fibroblasts. One LFS strain which immortalized, MDAH087 (087), had no detectable telomerase activity whereas another LFS strain which immortalized, MDAH041 (041), had detectable telomerase activity. In preimmortal cells from both strains, hTR was not detected by in situ hybridization. Immortal 087 cells remained negative for hTR, while immortal 041 cells demonstrated strong hTR in situ hybridization signals. 087 cells had long and heterogenous telomeres whereas telomeres of 041 cells had short, stable telomere lengths. Tumorigenicity studies in nude mice with ras-transformed 087 and 041 cells resulted in both cell lines giving rise to tumors and retaining telomerase status. Overall these results suggest that strain specificity may be important in telomerase re-activation and that both abrogation of p53 function and a mechanism to maintain telomeres are necessary for immortalization.

Expression of Wild-type p53 Is Required for Efficient Global Genomic Nucleotide Excision Repair in UV-irradiated Human Fibroblasts

We have shown previously that Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in the removal of UV-induced cyclobutane pyrimidine dimers from genomic DNA, but still proficient in the transcription-coupled repair pathway (Ford, J. M., and Hanawalt, P. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8876-8880). We have now utilized monoclonal antibodies specific for cyclobutane pyrimidine dimers or 6-4 photoproducts, respectively, to measure their repair in UV-irradiated human fibroblasts. Cells homozygous for p53 mutations were deficient in the repair of both photoproducts, whereas cells heterozygous for mutant p53 exhibited normal repair of 6-4 photoproducts, but decreased initial rates of removal of cyclobutane pyrimidine dimers, compared with normal cells. The specificity of the effect of wild-type p53 on nucleotide excision repair was demonstrated in a p53 homozygous mutant cell line containing a tetracycline-regulated wild-type p53 gene. Wild-type p53 expression and activity were suppressed in the presence of tetracycline, whereas withdrawal of tetracycline resulted in the induction of p53 expression, cell cycle checkpoint activation, and DNA damage-induced apoptosis. The regulated expression of wild-type p53 resulted in the recovery of normal levels of repair of both cyclobutane pyrimidine dimers and 6-4 photoproducts in genomic DNA, but did not alter the transcription-coupled repair of cyclobutane pyrimidine dimers. Therefore, the wild-type p53 gene product is an important determinant of nucleotide excision repair activity in human cells.

Cell cycle arrest defect in Li-Fraumeni Syndrome: a mechanism of cancer predisposition?

Cancer predisposition in approximately 60% of Li-Fraumeni Syndrome (LFS) families is associated with germline mutation of the TP53 gene. The p53 protein has been shown to mediate G1 arrest following DNA damage. We have investigated gamma-irradiation-induced transient and permanent G1 arrest in normal and LFS fibroblasts. The duration of transient G1 arrest varied between strains, but there was no difference in the range between normal (2-12 h) and LFS (1-13 h) cells. However, the extent of permanent G1 arrest was greatly reduced in LFS fibroblasts (mean 33+/-8% of the cell population) compared with normals (mean 67+/-9%) and correlated with their increased radiation survival (r=0.97, P<0.001). This phenotype was observed in LFS fibroblasts both with (seven cases) and without (two cases) TP53 mutation. Parallel studies with fibroblasts derived from cancer-prone, p53-deficient mice revealed no radiation-induced G1 cell cycle arrest in p53 null (-/-) cells. The p53 +/- cells were comparable to the wt p53 cells in transient G1 arrest capacity, but showed a diminished permanent G1 arrest. These data clearly implicate p53 function in permanent G1 arrest. The reduced capacity for DNA damage-induced, permanent G1 arrest in LFS may contribute significantly to cancer predisposition in this familial syndrome.

Role of transcription-coupled DNA repair in susceptibility to environmental carcinogenesis.

Susceptibility to environmental carcinogenesis is the consequence of a complex interplay between intrinsic hereditary factors and actual exposures to potential carcinogenic agents. We must learn the nature of these interactions as well as the genetic defects that confer enhanced risk. In some genetic diseases an increased cancer risk correlates with a defect in the repair or replications of damaged DNA. Examples include xeroderma pigmentosum (XP), ataxia telangiectasia, Fanconi's anemia, and Bloom's syndrome. In Cockayne's syndrome the Specific defect in transcription-coupled repair (TCR) does not predispose the patients to the sunlight-induced skin cancer characteristic of XP. The demonstration of TCR in the XP129 partial revertant of XP-A cells indicates that ultraviolet (UV) resistance correlates with repair of cyclobutane pyrimidine dimers in active genes. Repair measured as an average over the genome can be misleading, and it is necessary to consider genomic locations of DNA damage and repair for a meaningful assessment of the biological importance of particular DNA lesions. Mutations in the p53 tumor suppressor gene are found in many human tumors. TCR accounts for the resulting mutational spectra in the p53 gene in certain tumors. Li-Fraumeni syndrome fibroblasts expressing only mutant p53 are more UV-resistant and exhibit less UV-induced apoptosis than normal human cells or heterozygotes for mutations in only one allele of p53. The p53-defective cells are deficient in global excision repair capacity but have retained TCR. The loss of p53 function may lead to greater genomic instability by reducing the efficiency of global DNA repair while cellular resistance may be assured through the operation of TCR and the elimination of apoptosis.

Alterations in p53 and p16INK4 Expression and Telomere Length during Spontaneous Immortalization of Li-Fraumeni Syndrome Fibroblasts

Normal cells have a strictly limited growth potential and senesce after a defined number of population doublings (PDs). In contrast, tumor cells often exhibit an apparently unlimited proliferative potential and are termed immortalized. Although spontaneous immortalization of normal human cells in vitro is an extremely rare event, we observed this in fibroblasts from an affected member of a Li-Fraumeni syndrome kindred. The fibroblasts were heterozygous for a p53 mutation and underwent senescence as expected at PD 40. In four separate senescent cultures (A to D), there were cells that eventually recommenced proliferation. This was associated with aneuploidy in all four cultures and either loss (cultures A, C, and D) or mutation (culture B) of the wild-type (wt) p53 allele. Loss of wt p53 function was insufficient for immortalization, since cultures A, B, and D subsequently entered crisis from which they did not escape. Culture C has continued proliferating beyond 400 PDs and thus appears to be immortalized. In contrast to the other cultures, the immortalized cells have no detectable p16INK4 protein. A culture that had a limited extension of proliferative potential exhibited a progressive decrease in telomere length with increasing PD. In the culture that subsequently became immortalized, the same trend occurred until PD 73, after which there was a significant increase in the amount of telomeric DNA, despite the absence of telomerase activity. Immortalization of these cells thus appears to be associated with loss of wt p53 and p16INK4 expression and a novel mechanism for the elongation of telomeres.