Breast cancer and other neoplasms in women with neurofibromatosis type 1: a retrospective review of cases in the Detroit metropolitan area.

Neurofibromatosis type 1 (NF1) is one of the most common cancer predisposing syndromes with an incidence of 1 in 3,500 worldwide. Certain neoplasms or malignancies are over-represented in individuals with NF1; however, an increased risk of breast cancer has not been widely recognized or accepted. We identified 76 women with NF1 seen in the Henry Ford Health System (HFHS) from 1990 to 2009, and linked them to the Surveillance Epidemiology and End Results (SEER) registry covering the metropolitan Detroit area. Fifty-one women (67%) were under age 50 years at the time of data analysis. Six women developed invasive breast cancer before age 50, and three developed invasive breast cancer after age 50. Using standardized incidence ratios (SIRs) calculated based on the SEER age-adjusted invasive breast cancer incidence rates, our findings demonstrated a statistically significant increase of breast cancer incidence occurring in NF1 women (SIR = 5.2; 95% CI 2.4-9.8), and this relative increase was especially evident among those with breast cancer onset under age 50 (SIR = 8.8; 95% CI 3.2-19.2). These data are consistent with other reports suggesting an increase in breast cancer risk among females with NF1, which indicate that breast cancer screening guidelines should be evaluated for this potentially high-risk group.

Diverse system stresses: common mechanisms of chromosome fragmentation.

Chromosome fragmentation (C-Frag) is a newly identified MCD (mitotic cell death), distinct from apoptosis and MC (mitotic catastrophe). As different molecular mechanisms can induce C-Frag, we hypothesize that the general mechanism of its induction is a system response to cellular stress. A clear link between C-Frag and diverse system stresses generated from an array of molecular mechanisms is shown. Centrosome amplification, which is also linked to diverse mechanisms of stress, is shown to occur in association with C-Frag. This led to a new model showing that diverse stresses induce common, MCD. Specifically, different cellular stresses target the integral chromosomal machinery, leading to system instability and triggering of MCD by C-Frag. This model of stress-induced cell death is also applicable to other types of cell death. The current study solves the previously confusing relationship between the diverse molecular mechanisms of chromosome pulverization, suggesting that incomplete C-Frag could serve as the initial event responsible for forms of genome chaos including chromothripsis. In addition, multiple cell death types are shown to coexist with C-Frag and it is more dominant than apoptosis at lower drug concentrations. Together, this study suggests that cell death is a diverse group of highly heterogeneous events that are linked to stress-induced system instability and evolutionary potential.

Critical pathways in cellular senescence and immortalization revealed by gene expression profiling.

Bypassing cellular senescence and becoming immortal is a prerequisite step in the tumorigenic transformation of a cell. It has long been known that loss of a key tumor suppressor gene, such as p53, is necessary, but not sufficient, for spontaneous cellular immortalization. Therefore, there must be additional mutations and/or epigenetic alterations required for immortalization to occur. Early work on these processes included somatic cell genetic studies to estimate the number of senescence genes, and microcell-mediated transfer of chromosomes into immortalized cells to identify putative senescence-inducing genetic loci. These principal studies laid the foundation for the field of senescence/immortalization, but were labor intensive and the results were somewhat limited. The advent of gene expression profiling and bioinformatics analysis greatly facilitated the identification of genes and pathways that regulate cellular senescence/immortalization. In this review, we present the findings of several gene expression profiling studies and supporting functional data, where available. We identified universal genes regulating senescence/immortalization and found that the key regulator genes represented six pathways: the cell cycle pRB/p53, cytoskeletal, interferon-related, insulin growth factor-related, MAP kinase and oxidative stress pathway. The identification of the genes and pathways regulating senescence/immortalization could provide novel molecular targets for the treatment and/or prevention of cancer.

Genetic alteration of chromosome 8 is a common feature of human mammary epithelial cell lines transformed in vitro with benzo[a]pyrene.

While some epidemiological risk factors for breast cancer have been identified, the environmental factors responsible for transformation of mammary epithelial cells are not clear. We have exposed the spontaneously immortalized human mammary epithelial cell line MCF-10A to benzo[a]pyrene and selected transformed clones based on a loss of contact inhibition and anchorage-dependent growth. Cytogenetic studies showed that each of the transformed sublines possess an isochromosome 8q aberration. The c-Myc proto-oncogene, which is positioned at 8q24, was analyzed for changes in expression. Both c-Myc mRNA and protein levels were increased in the transformed clones relative to the parental cells. The transformed clones were not able to grow as tumors in vivo when injected into nude or SCID mice. To determine whether the involvement of chromosome 8 in BP-induced mutagenesis was a reproducible event, transformed clones were selected from three additional independently treated sets of BP-exposed MCF-10A cultures and analyzed by spectral karyotyping (SKY). These transformed sublines also harbored the isochromosome 8q abnormality. Data from this model show that benzo[a]pyrene, a ubiquitous procarcinogen, can induce selectable morphologic changes in a human mammary epithelial cell line, and that these transformed cells possess chromosomal aberrations frequently found in human breast tumors.

Visualization of the timing of gene amplification during multistep head and neck tumorigenesis.

Head and neck tumorigenesis is thought to represent a multistep process whereby carcinogen exposure leads to genetic instability in the tissue and the accumulation of specific genetic events, which result in dysregulation of proliferation, differentiation, and cell loss and the acquisition of invasive capacity. Chromosome 11q13 amplification is frequently observed in head and neck squamous cell carcinoma (HNSCC), and the amplified gene products are assumed to play important functional roles in the tumor phenotype. However, it is not well understood whether gene amplification precedes carcinoma development or results from the unstable nature of intact tumors. To determine the timing of gene amplification during tumorigenesis, tissue sections from amplified HNSCC specimens (containing a contiguous transition from normal epithelium to hyperplasia to dysplasia to carcinoma) were probed for INT2 gene copy number by chromosome in situ hybridization. In addition, representative epithelia were microdissected from the tissue sections, and the DNA was isolated and assessed for INT2 gene copy number by semiquantitative PCR. In those cases containing amplified INT2 in the carcinoma, gene amplification appeared to precede HNSCC development. In one case, INT2 gene amplification appeared in the hyperplasia to dysplasia transition, whereas in two other cases, gene amplification was apparent at dysplasia. These results suggest that gene amplification can occur early during head and neck tumorigenesis and that genetic instability is an important driving force in the tumorigenesis process.

PolyADP-ribose polymerase is a coactivator for AP-2-mediated transcriptional activation.

Overexpression of transcription factor AP-2 has been implicated in the tumorigenicity of the human teratocarcinoma cell lines PA-1 that contain an activated ras oncogene. Here we show evidence that overexpression of AP-2 sequesters transcriptional coactivators which results in self-inhibition. We identified AP-2-interacting proteins and determined whether these proteins were coactivators for AP-2-mediated transcription. One such interacting protein is polyADP-ribose polymerase (PARP). PARP suppresses AP-2 self-inhibition and enhances AP-2 activity in PA-1 cells indicating that it is a coactivator for AP-2-transcription. PARP significantly restores AP-2 transcriptional activity in ras oncogene-transformed cells suggesting that it might suppress transformation in these cells. Another AP-2-interacting protein, RAP74, a subunit of transcription factor TFIIF, does not affect AP-2-mediated transcriptional activation alone or in the presence of RAP30, the other subunit of TFIIF. RAP74 also fails to relieve AP-2-mediated transcriptional self-interference and cross-interference. These studies suggest that the interaction between AP-2 and RAP74 may have functions other than activation of AP-2-mediated transcription.

Coactivator PC4 mediates AP-2 transcriptional activity and suppresses ras-induced transformation dependent on AP-2 transcriptional interference.

ras oncogene-transformed PA-1 human teratocarcinoma cells have abundant AP-2 mRNA but, paradoxically, little AP-2 transcriptional activity. We have previously shown that overexpression of AP-2 in nontumorigenic variants of PA-1 cells results in inhibition of AP-2 activity and induction of tumorigenicity similar to that caused by ras transformation of PA-1 cells. Evidence indicated the existence of a novel mechanism of inhibition of AP-2 activity involving sequestering of transcriptional coactivators. In this study, we found that PC4 is a positive coactivator of AP-2 and can restore AP-2 activity in ras-transformed PA-1 cells. Relative to vector-transfected ras cell lines, ras cell lines stably transfected with and expressing the PC4 cDNA have a diminished growth rate and exhibit a loss of anchorage-independent growth, and they are unable to induce the formation of tumors in nude mice. These data suggest that a transcriptional coactivator, like a tumor suppressor, can have a growth-suppressive effect on cells. Our experiments are the first to show that ras oncogenes and oncogenic transcription factors can induce transformation through effects on the transcription machinery rather than through specific programs of gene expression.

Cloning and characterization of the Drosophila homologue of the AP-2 transcription factor.

The AP-2 family of transcription factors (AP-2alpha, AP-2beta and AP-2gamma) is temporally and spatially regulated in mammals and has also been implicated in oncogenesis. Here we report the isolation of genomic and cDNA clones encoding the Drosophila homologue of AP-2, designated DAP-2. The predicted amino acid sequence exhibits 42-45% overall identity with the vertebrate AP-2 proteins. A sequence of 107 amino acids within the DNA binding and dimerization domain of the vertebrate AP-2 proteins is highly conserved (90-92%) with the DAP-2 homologue. An in vitro translation product of DAP-2 cDNA binds specifically to AP-2 consensus binding sites. DAP-2 was also shown to be functionally conserved in vivo because transient transfection of a DAP-2 expression plasmid activated transcription through AP-2 binding sites in both mammalian and Drosophila cell lines. DAP-2 is expressed during early embryogenesis and DAP-2 transcripts are also detected in the adult. Whole-mount in situ hybridizations demonstrated that DAP-2 is expressed initially at stage 9 of Drosophila embryonic development and that DAP-2 transcripts are detected in regions of the brain, eye-antennal disc, optical lobe, antenno-maxillary complex, and in a subset of cells of the ventral nerve cord. The cloning of DAP-2 and the identification of the DAP-2 expression pattern during embryogenesis provides a starting point to address the function of AP-2 during differentiation and development in a well understood model system.

Loss of AP-2 results in downregulation of c-KIT and enhancement of melanoma tumorigenicity and metastasis.

Expression of the tyrosine kinase receptor, c-KIT, progressively decreases during local tumor growth and invasion of human melanomas. We have previously shown that enforced c-KIT expression in highly metastatic cells inhibited tumor growth and metastasis in nude mice. Furthermore, the ligand for c-KIT, SCF, induces apoptosis in human melanoma cells expressing c-KIT under both in vitro and in vivo conditions. Here we show that loss of c-KIT expression in highly metastatic cells correlates with loss of expression of the transcription factor AP-2. The c-KIT promoter contains three binding sites for AP-2 and EMSA gels demonstrated that AP-2 protein binds directly to the c-KIT promoter. Transfection of wild-type AP-2 into c-KIT-negative A375SM melanoma cells activated a c-KIT promoter-driven luciferase reporter gene, while expression of a dominant-negative AP-2B in c-KIT-positive Mel-501 cells inhibited its activation. Endogenous c-KIT mRNA and expression of proteins were upregulated in AP-2-transfected cells, but not in control cells. In addition, re-expression of AP-2 in A375SM cells suppressed their tumorigenicity and metastatic potential in nude mice. These results indicate that the expression of c-KIT is highly regulated by AP-2 and that enforced AP-2 expression suppresses tumorigenicity and metastatic potential of human melanoma cells, possibly through c-KIT transactivation and SCF-induced apoptosis. Therefore, loss of AP-2 expression might be a crucial event in the development of malignant melanoma.

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.

pZ402, an improved SV40-based shuttle vector containing a T-antigen mutant unable to interact with wild-type p53.

Shuttle vectors are useful tools for studying DNA replication and mutagenesis. SV40-based shuttle vectors are popular because of their ease of use and quick results. However, one complication with the use of SV40-based shuttle vectors is the interaction of cellular p53 protein with the T-antigen of SV40. Wild-type, but not mutant p53 has been shown to be involved in DNA replication and DNA repair. To address this concern, we have modified an SV40-based shuttle vector, pZ189, by exchanging the wt T-antigen for a mutant SV40 T-antigen, which is unable to bind with p53. This shuttle vector, pZ402, provides us with a tool to study DNA replication and genomic instability in cells with varying genetic backgrounds without interference from the interaction of T-antigen with p53.

Loss of AP-2 results in up-regulation of MCAM/MUC18 and an increase in tumor growth and metastasis of human melanoma cells.

MCAM/MUC18 is a cell-surface glycoprotein of 113 kDa, originally identified as a melanoma antigen, whose expression is associated with tumor progression and the development of metastatic potential. We have previously shown that enforced expression of MCAM/MUC18 in primary cutaneous melanoma led to increased tumor growth and metastatic potential in nude mice. The mechanism for up-regulation of MCAM/MUC18 during melanoma progression is unknown. Here we show that up-regulation of MCAM/MUC18 expression in highly metastatic cells correlates with loss of expression of the transcription factor AP-2. The MCAM/MUC18 promoter contains four binding sites for AP-2, and electrophoretic mobility shift assay gels demonstrated that the AP-2 protein bound directly to the MCAM/MUC18 promoter. Transfection of AP-2 into highly metastatic A375SM melanoma cells (AP-2-negative and MCAM/MUC18-positive) inhibited MCAM/MUC18 promoter-driven chloramphenicol acetyltransferase reporter gene in a dose-dependent manner. MCAM/MUC18 mRNA and protein expression were down-regulated in AP-2-transfected but not in control cells. In addition, re-expression of AP-2 in A375SM cells inhibited their tumorigenicity and metastatic potential in nude mice. These results indicate that the expression of MCAM/MUC18 is regulated by AP-2 and that enforced AP-2 expression suppresses tumorigenicity and metastatic potential of human melanoma cells, possibly by down-regulating MCAM/MUC18 gene expression. Since AP-2 also regulates other genes that are involved in the progression of human melanoma such as c-KIT, E-cadherin, MMP-2, and p21(WAF-1), we propose that loss of AP-2 is a crucial event in the development of malignant melanoma.

drp, a novel protein expressed at high cell density but not during growth arrest.

Contact is a vital mechanism used by cells to interact with their environment. Contact with living and nonliving elements adjacent to a cell is the basis for many common biological events ranging from growth regulation to metastasis to embryonic pattern formation. We describe the cloning and characterization of a novel density-regulated protein (drp) whose expression is increased in cultured cells at high density compared with cells at low density. A drp cDNA was isolated from the human teratocarcinoma cell line PA-1. Northern analysis with a drp probe revealed transcripts of 2.8 and 3.2 kb. The drp RNA was expressed in a variety of tissues, with the highest amounts in skeletal and cardiac muscle. Using antipeptide antisera, increasing amounts of a 70-kDa protein were detected using several experimental approaches in several cells lines as cell density is increased. Conditioned medium from high-density cells was unable to induce expression of drp in cells growing at low density. Similarly, growth arrest by serum starvation or transforming growth factor-beta (TGF-beta) treatment failed to elicit drp expression. We conclude that drp is a novel protein whose expression is increased at high cell density but not growth arrest.

Enhanced Krev-1 expression inhibits the growth of pancreatic adenocarcinoma cells.

Pancreatic ductal adenocarcinoma is characterized by a high rate of activating mutations involving codon 12 of the K-ras protooncogene. As a means of ras-targeted intervention, the effects of enhanced Krev-1 gene expression on the growth and tumorigenicity of the hamster pancreatic adenocarcinoma cell line PC-1 were evaluated. Overexpression of the Krev-1 gene product resulted in morphologic reversion to a less transformed phenotype, as well as retarded growth kinetics and diminished potential for anchorage-independent growth. Among six transfected cell lines, the magnitude of these changes correlated with the degree of Krev-1 overexpression as assessed by Western blot. When PC-1 cells overexpressing high levels of the Krev-1 gene product were assessed for tumorigenicity in syngeneic animals, an increased latency to tumor growth and a decreased tumor size were noted. The results confirm that overexpression of the Krev-1 gene may provide a useful strategy for ras-targeted intervention in this disease.

Functional interaction between a RARE and an AP-2 binding site in the regulation of the human HOX A4 gene promoter.

HOX A genes are induced in a temporal fashion after retinoic acid (RA) treatment in non-N-ras-transformed PA-1 human teratcarcinoma cells. However, In N-ras-transformed PA-1 cells, RA-Induced expression of HOX A genes is delayed. The mRNA for the transcriptional activator AP-2 is overexpressed in these ras-transformed cells, but AP-2 transcriptional activity is inhibited relative to non ras-transformed PA-1 cells. Constitutive expression of AP-2 mimics the effect of ras by transforming cells and inhibiting differentiation in culture. We analyzed 4 kb of the human HOX A4 gene promoter and identified seven putative AP-2-binding sites in the DNA sequence. Transcription assays with variably sized HOX A4 promoter reporter constructs revealed that a 365 bp region of the promoter, -2950 to -3315 relative to the mRNA start, controls RA responsiveness and ras-mediated inhibition of HOX A4 activity. This region contains an AP-2 binding site and a RARE. Elimination of the AP-2 site by site-directed mutagenesis demonstrated that the AP-2 site is involved in RA-mediated transcriptional activation of the human HOX A4 promoter in combination with the RA receptor response element (RARE). In N-ras-transformed cells, low HOX A4 promoter activity results from ras inhibition of AP-2 transactivation.

Analysis of genomic instability in Li-Fraumeni fibroblasts with germline p53 mutations.

Germline p53 mutations are frequently observed in the normal DNA of cancer-prone patients with Li-Fraumeni syndrome (LFS). Fibroblasts from LFS patients develop chromosomal aberrations, loss of cell cycle control, and spontaneous immortalization. We transfected four different mutant p53 genes into human skin fibroblasts from normal donors with two copies of wild-type p53 (p53(wt/wt)). Each mutant p53 expression-plasmid induced genomic instability equivalent to that seen in LFS cells. To test the role of wild-type and mutant p53 alleles in DNA replication and fidelity in LFS cells, we analysed the replication of the SV40-based shuttle vector pZ189 in four types of cells. We used p53(wt/mut) and p53(mut/-) LFS fibroblasts, and p53(-/-) non-LFS cells. Replication of pZ189 in vivo was significantly reduced by the presence of a p53(wt) allele. To show that this was not just due to inhibition of the function of T-antigen in SV40-based replication, we constructed a shuttle vector, pZ402, that contains a mutation in SV40 T-antigen which blocks its ability to interact with p53. Replication of pZ402 in LFS cells was also reduced by the presence of p53(wt), indicating that p53 can inhibit replication by interacting with proteins within the cellular replication machinery. Replicative errors in this shuttle vector are detected as mutations in a marker gene, supF. In addition to supF mutations, we observed deletion of a portion of the SV40 T-antigen gene in 100% of replicated plasmid pZ189 mutants (supF-) from the p53(wt/mut) fibroblasts and in 88% of the supF mutants from the p53(mut/-) (amino acid 175 arg to his) LFS cells. In one cell strain of immortal LFS cells, P53(mut/-) , containing a p53 frameshift mutation at amino acid 184, pZ189 replication yielded very few of these deleted shuttle vector plasmids (15%). These large deletions were not detected in plasmids replicated in p53(-/-) non-LFS cells, Saos-2 cells. Replicated plasmids with a normal supF gene were never found to have this large deletion regardless of the cell from which they were derived. Because the supF gene is not in the same region of the shuttle vector as the T-antigen gene it appears that second, independent gene deletions are frequent when replicative errors in supF occur in cells with a mutant p53. We conclude, therefore, that p53(wt/mut) LFS cells contain an activity that promotes mutations. Such an activity, which is likely to be due to the p53(mut), could result in the high rate of chromosomal instability and allelic loss of the wild-type p53 observed as these cells spontaneously immortalize.

Coordinate control of growth and cytokeratin 13 expression by retinoic acid.

Retinoic acid (RA) modulates the growth and differentiation of various normal and malignant cells. These effects are most likely mediated by changes in gene expression. Genes whose expression is modulated by RA may be useful as markers of growth responsiveness to retinoids. Using differential cDNA cloning we identified 10 genes regulated by RA in the head and neck squamous cell carcinoma cell line MDA886Ln. Keratin (K) 13 gene expression was the gene expression most related to the degree of sensitivity of growth to RA, as K13 was not expressed in a series of RA-resistant cell lines. Our data suggest that low K13 expression may be mechanistically related to resistance to RA-induced growth inhibition.

Predominance of the metastatic phenotype in hybrids formed by fusion of mouse and human melanoma clones.

The fusion of mouse and human melanoma cells that were tumorigenic but had different metastatic capabilities resulted in hybrids that were metastatic when injected intravenously or subcutaneously into nude mice, regardless of whether it was the mouse or the human melanoma clone that was metastatic. The H7 hybrid line, formed by fusing murine nonmetastatic K1735 C19 cells with human metastatic A375 C15 cells retained high metastatic potential over more than 50 sub-culture passages, suggesting that the dominant metastatic phenotype in these hybrid cells was stable. Using fluorescent in situ hybridization (FISH), human chromosome 17 was consistently identified as the predominant human chromosome in the majority of H7 cells tested between passages 20 and 60. Western blot analysis showed that the hybrid cells expressed human nm23 protein, indicating that at least one gene on the human chromosome 17 was functional. Immunocytochemistry and immunoprecipitation showed that the metastatic A375 C15 and H7 cells expressed p53 protein, but that the nonmetastatic K1735 C19 melanoma cells did not. Sequencing the human p53 gene in A375 C15N and H7 showed mutations in exon 7. Using a bioassay technique, we showed that K1735 C19 cells can spread from subcutaneous tumors to the lungs of nude mice yet fail to form metastases. With the addition of human chromosome 17 from A375 C15 cells, which carries a mutant p53 gene, the cells readily formed lung metastases. In this melanoma hybrid, a mutant p53 gene appears to confer a survival advantage on cells arrested in the lungs of nude mice and thus contributes to the growth of metastatic cells.

Structure and function of the HOX A1 human homeobox gene cDNA.

Homeobox genes code for transcription factors and are arranged in clusters, named A, B, C and D, found on four separate chromosomes in vertebrates. They contain a homeobox DNA sequence which codes for the homeodomain, a region of amino acids responsible for the DNA binding exhibited by these proteins. During embryonic development, the homeobox genes are both spatially and temporally regulated. In teratocarcinoma cell cultures, homeobox genes are regulated by retinoic acid (RA). The cDNAs from the first gene in the human HOX A cluster, HOX A1 (1.6), were cloned and the nucleotide sequence of a full-length cDNA was determined. It is highly homologous to its murine counterpart. Another HOX A1 cDNA was cloned, corresponding to an alternatively spliced form. In vitro translation of the full-length cDNA clone gave rise to a protein of 36 kDa. In PA-1 human teratocarcinoma cells HOX A1 is the earliest HOX A gene to be expressed after treatment with RA. To test whether HOX A1 could function as a early regulator of other HOX A cluster genes, we cotransfected into PA-1 human teratocarcinoma cells sense and antisense HOX A1 cDNAs expressed from an SV40 promoter with a 5.4-kb RA-sensitive HOX A4 (1.4) promoter-cat reporter. We found no effect of HOX A1 on the HOX A4 promoter. However, cotransfection of HOX A5 (1.3) was able to inhibit the HOX A4 promoter activity.

Defective G2 checkpoint function in cells from individuals with familial cancer syndromes.

The early events in the G2 checkpoint response to ionizing radiation (IR) were analyzed in diploid normal human fibroblasts (NHFs) and fibroblasts from patients with two heritable cancer syndromes. Exposure to gamma-radiation of asynchronously growing NHFs resulted in a rapid reduction in the number of cells in mitosis (G2 delay) and was accompanied by a quantitatively similar reduction in the p34CDC2/cyclin B in vitro histone H1 kinase activity as compared with sham-treated controls. This G2 delay was strong by 1 h following exposure to IR, maximal by 2 h, and was accompanied by an accumulation of tyrosine-phosphorylated p34CDC2 molecules. In contrast, fibroblasts from individuals with ataxia telangiectasia displayed significantly less reduction of the mitotic index or histone H1 kinase activity after IR. Low passage fibroblasts from individuals with Li-Fraumeni syndrome having one wild-type and one mutated p53 allele were similar to NHFs in their immediate G2 checkpoint response to IR, as were NHFs expressing the human papilloma virus type 16 E6 gene product (functionally inactivating p53) and low passage cells from p53-deficient mouse embryos. However, the p53-deficient fibroblasts were genomically unstable and became defective in their early G2 checkpoint response to IR. Furthermore, immortal Li-Fraumeni syndrome fibroblasts lacking wild-type p53 displayed an attenuated G2 checkpoint response. These results link the early events in G2 checkpoint response to IR in NHFs with a rapid inhibition of p34CDC2/cyclin B protein kinase activity and demonstrate that while not required for this immediate G2 delay, lack of p53 can lead to subsequent genetic alterations that result in defective G2 checkpoint function.