Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase.

Whether Chk2 contributes to DNA damage-induced arrest in G2 has been controversial. To investigate this issue further, we generated Chk2-deficient DT40 B-lymphoma cells by gene targeting and compared their cell cycle response to ionizing radiation (IR) with wild-type (WT) and isogenic Chk1-deficient counterparts. After moderate doses of IR (4 Gy), we find that Chk2-/- cells which are in G1 or S phase at the time of irradiation arrest efficiently in G2. In contrast, Chk2-/- cells which are in G2 when DNA damage is incurred exhibit an impaired mitotic delay compared to WT, with the result that cells enter mitosis with damaged DNA as judged by the presence of numerous gamma-H2AX foci on condensed chromosomes. Impaired G2 delay as the result of Chk2 deficiency can be detected at very low doses of radiation (0.1 Gy), and may allow division with spontaneous DNA damage, since a higher proportion of mitotic Chk2-/- cells bear spontaneous gamma-H2AX foci and damaged chromosomes during unperturbed growth compared to WT. The contribution of Chk2 to G2/M delay is epistatic to that of Chk1, since Chk1-/- cells exhibit no measurable mitotic delay at any radiation dose tested. We suggest that this function of Chk2 could contribute to tumour suppression, since cell division with low levels of spontaneous damage is likely to promote genetic instability and thus carcinogenesis.

Chk1-dependent slowing of S-phase progression protects DT40 B-lymphoma cells against killing by the nucleoside analogue 5-fluorouracil.

Chk1 plays a crucial role in the DNA damage and replication checkpoints in vertebrates and may therefore be an important determinant of tumour cell responses to genotoxic anticancer drugs. To evaluate this concept we compared the effects of the nucleoside analogue 5-fluorouracil (5FU) on cell cycle progression and clonogenic survival in DT40 B-lymphoma cells with an isogenic mutant derivative in which Chk1 function was ablated by gene targeting. We show that 5FU activates Chk1 in wild-type DT40 cells and that 5FU-treated cells accumulate in the S phase of the cell cycle due to slowing of the overall rate of DNA replication. In marked contrast, Chk1-deficient DT40 cells fail to slow DNA replication upon initial exposure to 5FU, despite equivalent inhibition of the target enzyme thymidylate synthase, and instead accumulate progressively in the G1 phase of the following cell cycle. This G1 accumulation cannot be reversed rapidly by exogenous thymidine or removal of 5FU, and is associated with increased incorporation of 5FU into genomic DNA and severely diminished clonogenic survival. Taken together, these results demonstrate that a Chk1-dependent replication checkpoint which slows S phase progression can protect tumour cells against the cytotoxic effects of 5FU.

Herpes simplex virus type 1 blocks the apoptotic host cell defense mechanisms that target Bcl-2 and manipulates activation of p38 mitogen-activated protein kinase to improve viral replication.

Wild-type (wt) herpes simplex virus type 1 (HSV-1) suppresses cell death. We investigated the apoptotic pathways triggered during infection with mutant viruses tsk and 27lacZ (which lack functional ICP4 and ICP27 viral proteins, respectively) and examined the mechanisms used by wt HSV-1 to protect against programmed cell death induced by the DNA-damaging compound cisplatin. In our studies, we used BHK and HeLa cells, with similar results. We suggest that a decrease in the levels of Bcl-2 protein is a key event during apoptosis induced by the mutant viruses and that Bcl-2 levels are targeted by (i) a decrease of bcl-2 RNA, (ii) caspase-related proteolysis, and (iii) p38 mitogen-activated protein kinase (p38MAPK)-dependent destabilization of Bcl-2 protein. We show that wt HSV-1, but not the mutant viruses, maintains bcl-2 RNA and protein levels during infection and protects from the cisplatin-induced decrease in bcl-2 RNA; our data suggest that both ICP27 and ICP4 are required for this function. Additionally, wt HSV-1 evades but does not actively block activation of caspases. Although wt HSV-1 induces p38MAPK activation during infection, it prevents p38MAPK-dependent destabilization of Bcl-2 and exploits p38MAPK stimulation to enhance transcription of specific viral gene promoters to increase viral yields.

Allelic loss at the BRCA1, BRCA2 and TP53 loci in human sporadic breast carcinoma.

Abnormalities in several genes are known to confer susceptibility to breast cancer. In the present study, we investigated the incidence of allelic imbalance at the BRCA1, BRCA2 and TP53 loci, in 82 sporadic breast carcinomas using a bank of highly polymorphic microsatellite markers located at the BRCA1, BRCA2 and TP53 regions. Genetic alterations were observed in 58/82 (71%) cases in at least one microsatellite marker, at one of the three regions. Twenty-seven out of 82 (33%) cases exhibited loss of heterozygosity (LOH) at BRCA1 locus while in 20/82 (34%) cases LOH was observed for the BRCA2 region. Allelic deletions were detected in 28/82 (34%) cases for the TP53 locus. Our results suggest that allelic deletion at the above genetic loci play an important role to the development of sporadic breast tumours.

Herpes simplex virus type 1 infection stimulates p38/c-Jun N-terminal mitogen-activated protein kinase pathways and activates transcription factor AP-1.

Cells respond to environmental stress and proinflammatory cytokines by stimulating the Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and the p38 mitogen-activated protein kinase cascades. Infection of eukaryotic cells with herpes simplex virus type 1 (HSV-1) resulted in stimulation of both JNK/SAPK and p38 mitogen-activated protein kinase after 3 h of infection, and activation reached a maximum of 4-fold by 9 h post-infection. By using a series of mutant viruses, we showed that the virion transactivator protein VP16 stimulates p38/JNK, whereas no immediate-early, early, or late viral expressed gene is involved. We identified the stress-activated protein kinase kinase 1 as an upstream activator of p38/JNK, and we demonstrated that activation of AP-1 binding proceeded p38/JNK stimulation. During infection, the activated AP-1 consisted mainly of JunB and JunD with a simultaneous decrease in the cellular levels of Jun protein. We suggest that activation of the stress pathways by HSV-1 infection either represents a cascade triggered by the virus to facilitate the lytic cycle or a defense mechanism of the host cell against virus invasion.

Transcriptional regulation of the c-H-ras1 gene by the P53 protein is implicated in the development of human endometrial and ovarian tumours.

The human c-H-ras1 gene contains within the first intron a p53 element, which functions as a transcriptional enhancer. Using nuclear extracts from human endometrial and ovarian tumours in gel retardation assays, we examined the binding levels of the P53 protein to the H-ras element in tumour versus the adjacent normal tissue. Elevated P53 binding in the tumour tissue was found in 5/12 (42%) endometrial and in 2/5 (40%) ovarian specimens and these cases were found to overexpress wild-type P53. Loss of P53 binding to the H-ras element due to p53 mutations, was observed in 3/12 (25%) endometrial and in 1/5 (20%) ovarian cases. Similar P53 binding levels to the H-ras element were found in 4/12 (33%) endometrial and in 2/5 (40%) ovarian pairs showing normal expression of wild-type P53. Overexpression of the Ras p21 protein correlated with elevated binding and increased nuclear levels of wild-type P53. Our results suggest that P53 protein alterations, participate in the development of human gynecological neoplasias through aberrant transcriptional regulation of the H-ras proto-oncogene.

Evidence for loss of heterozygosity in human psoriatic lesions.

Psoriasis, a disease of human skin, is characterized by abnormal differentiation and hyperproliferation of keratinocytes; it has a genetic background. Using 11 highly polymorphic microsatellite markers on eight chromosome arms, we performed an allelotype analysis in 14 psoriatic plaques, in order to reveal any chromosome deletions involved in the development of the disease. We detected loss of heterozygosity (LOH) on at least one microsatellite marker in nine of 14 (64%) cases. We also observed particular genetic loci altered with LOH, on chromosomes 3p, 7p/q and 8p. Our results suggest that LOH is an important phenomenon in the development of psoriatic plaques, providing evidence for deletion of regulatory genes.

Endometrial carcinoma in a breast cancer patient treated with tamoxifen.

The use of tamoxifen for breast cancer therapy is linked to an increased danger of developing endometrial neoplasias in postmenopausal patients receiving the drug. Understanding the molecular mechanisms of the tumorigenic activity of tamoxifen may be of great prognostic and therapeutic significance. Our study suggests that tamoxifen treatment and alterations of the K-ras proto-oncogene may occur as parallel events in carcinogenesis of the endometrium.

The association of the H-ras oncogene and steroid hormone receptors in gynecological cancer.

Expression of the ras family of cellular oncogenes is associated with tumorigenicity, invasiveness and metastatic potential in a variety of human carcinomas. Additionally, H-ras cooperates with glucocorticoids and with ovarian hormones in cell transformation and in the development of mammary carcinomas. Steroids are considered to be tumor promoters and their levels influence the cure rates and survival of the patients with gynecological lesions. It is proposed that they exert tumor promoting activity by transcriptional regulation of nuclear proto-oncogenes, such as c-fos, c-jun, and c-myc. The human H-ras gene contains within its first and fourth introns, sequences that are specifically recognized by glucocorticoid and estrogen receptors, respectively. Using gel retardation assays, the level of steroid receptor binding in H-ras elements has been compared, employing nuclear extracts from human endometrial and ovarian lesions and from the adjacent normal tissue. Elevated binding of the glucocorticoid and estrogen receptors in the corresponding H-ras elements in almost all tissue pairs tested has been found. It is suggested that the H-ras proto-oncogene is hormonally regulated and directly implicated in human gynecological cancer through elevated, steroid-induced gene expression.

Glucocorticoid and estrogen receptors have elevated activity in human endometrial and ovarian tumors as compared to the adjacent normal tissues and recognize sequence elements of the H-ras proto-oncogene.

We examined the level of receptor binding in H-ras elements, using nuclear extracts derived from human endometrial and ovarian lesions and from adjacent normal tissue in gel retardation assays. We found increased binding of the glucocorticoid receptor (GR) to the H-ras GR element in more than 90% of endometrial tumors and in all ovarian tumors tested, as compared to the corresponding adjacent normal tissue. Additionally, we found elevated binding of the estrogen receptor (ER) in H-ras ER element in all pairs of ovarian tumor/normal tissue tested, whereas in ER-negative control breast tumor/normal tissue pairs, no differences in ER DNA-binding levels were observed. These results suggest that steroid hormone receptor binding could directly activate the H-ras oncogenic potency in human endometrial and ovarian lesions, providing additional evidence for the role of H-ras expression in hormonally responsive human cancers.

Binding of wild-type and mutant forms of p53 protein from human tumors to a specific DNA-sequence of the first intron of the h-ras oncogene.

p53 is the most frequent target for genetic alterations in a wide variety of human cancers. The product of the p53 tumor suppressor gene binds to DNA and activates transcription from promoters containing its consensus binding site. In the accompanying paper we have found that P53 tumor suppressor protein recognizes specifically a transcriptional element within the human H-ras protooncogene (Spandidos DA, et al, Int J Oncol 7: 1029-1034, 1995). We transfected Saos-2 cells, which are p53-null cells, with plasmids encoding for the wild type (wt) and for one 'hot spot' mutant (mt) of the p53 gene (H 273). Using the resulted nuclear extracts for gel retardation assays, we showed binding of both the wild-type and the mutant form of p53 to the H-ras DNA. Furthermore, using nuclear extracts from head and neck tumors and from adjacent normal tissues in gel retardation assays, we found binding of both the wild-type and the p53 mutant in the same responsive element of the H-ras oncogene. These experimental results suggest a direct role of p53 in regulation of H-ras. Identification of cellular proto-oncogenes as mediators of the transcriptional effects of wild-type and mutant forms of p53 gene, will be a step towards a better understanding of the role of oncogenes and once-suppressor genes in tumor promotion.

Specific recognition of a transcriptional element within the human h-ras protooncogene by the p53 tumor-suppressor.

The nuclear phosphoprotein p53 is frequently inactivated in human cancer. Although it was previously classified as an oncoprotein, p53 has emerged as a tumor suppressor controlling cell cycle progression by regulating gene transcription. A major biochemical property of wild-type p53 is its ability to bind DNA in a sequence-specific manner. The human c-H-ras gene contains within its first intron sequences that partially match the p53 consensus binding site. We determined that these sequences represent a bona fide p53 element, since in vitro translated wild-type p53 recognized them with high affinity. Furthermore, wild-type p53 activated transcription from a reporter plasmid containing the c-H-ras element as an enhancer. These findings suggest that p53 regulates cellular growth by coordinate transcription of genes that suppress and promote cellular proliferation.

Binding of the glucocorticoid and estrogen-receptors to the human h-ras oncogene sequences.

There is evidence that hormone regulation of cellular oncogenes plays an essential role in human cancer. The c-H-ras gene is implicated through both mutation and abnormal gene expression in many types of human cancer. Computer scanning of this gene has revealed two putative hormone response motifs: A possible Glucocorticoid Response Element (GRE) at position 1261 of the first intron of the H-ras1 gene and a putative Estrogen Response Element (ERE), at position 3007 of the fourth intron of the gene. In DNA binding assays, using the HeLa and LATK-cell lines, we showed specific binding of the corresponding receptors at both putative H-ras glucocorticoid and estrogen response sequences, suggesting that hormones could be contributing to H-ras transcriptional regulation through interaction with their corresponding Hormone Response Elements (HREs).