Involvement of Schizosaccharomyces pombe Srs2 in cellular responses to DNA damage.

In the budding yeast Saccharomyces cerevisiae the Srs2/RadH DNA helicase promotes survival after ultraviolet (UV) irradiation, and has been implicated in DNA repair, recombination and checkpoint signalling following DNA damage. A second helicase, Sgs1, is the S.cerevisiae homologue of the human BLM and WRN proteins, which are defective in cancer predisposition and/or premature ageing syndromes. Saccharomyces cerevisiae cells lacking both Srs2 and Sgs1 exhibit a severe growth defect. We have identified an Srs2 orthologue in the fission yeast Schizosaccharomyces pombe, and have investigated its role in responses to UV irradiation and inhibition of DNA replication. Deletion of fission yeast srs2 caused spontaneous hyper-recombination and UV sensitivity, and simultaneous deletion of the SGS1 homologue rqh1 caused a severe growth defect reminiscent of that seen in the equivalent S.cerevisiae mutant. However, unlike in budding yeast, inactivation of the homologous recombination pathway did not suppress this growth defect. Indeed, the homologous recombination pathway was required for maintenance of normal fission yeast viability in the absence of Srs2, and loss of homologous recombination and loss of Srs2 contributed additively to UV sensitivity. We conclude that Srs2 plays related, but not identical, roles in the two yeast species.

Genetic disorders associated with cancer predisposition and genomic instability.

Genomic instability in its broadest sense is a feature of virtually all neoplastic cells. In addition to the mutations and/or gene amplifications that appear to be a prerequisite for the acquisition of a neoplastic phenotype, human cancers exhibit other "markers" of genomic instability--in particular, a high degree of aneuploidy. Indeed, many studies have shown that aneuploidy is an almost invariant feature of cancer cells, and it has been argued by some that the emergence of aneuploid cells is a necessary step during tumorigenesis. The functional link between genomic instability and cancer is strengthened by the existence of several "genetic instability" disorders of humans that are associated with a moderate to severe increase in the incidence of cancers. These disorders include ataxia telangiectasia, Bloom's syndrome, Fanconi anemia, xeroderma pigmentosum, and Nijmegen breakage syndrome, all of which are very rare and are inherited in a recessive manner. Analysis of the cells from such cancer-prone individuals is clearly a potentially fruitful approach for delineating the genetic basis for instability in the genome. It is assumed that by identifying the underlying cause of genetic instability in these disorders, one can derive valuable information not only about the basis of particular genetic diseases, but also about the underlying causes of genomic instability in sporadic cancers in the general population. In this article, we review the clinical and cellular properties of genetic instability disorders associated with cancer predisposition. In particular, we focus on the rapid advances made in our understanding of these disorders that have derived from the cloning of the genes mutated in each case. Because in many instances the affected genes have analogs in lower eukaryotic species, we shall discuss how studies in yeasts in particular have proved valuable in our understanding of human diseases and predisposition to cancer.

p53 regulates Cdc2 independently of inhibitory phosphorylation to reinforce radiation-induced G2 arrest in human cells.

We have investigated the influence of p53 on radiation-induced G2 cell cycle arrest using human H1299 cells expressing temperature-sensitive p53. Gamma-irradiated cells lacking p53 arrested transiently in G2 with Cdc2 extensively phosphorylated at the inhibitory sites Thr14 and Tyr15, and with both Cdc2 and cyclin B1 restricted to the cytoplasm. Activation of p53 by temperature shift resulted in a more protracted G2 arrest that could not be overridden by checkpoint-abrogating drugs. Surprisingly, this enhancement of G2 arrest was associated with a marked lack of inhibitory phosphorylation of Cdc2 and with the nuclear localization of both Cdc2 and cyclin B1. While transient expression of an A14F15 mutant form of Cdc2 that is not subject to inhibitory phosphorylation induced mitotic catastrophe in cells lacking p53, the p53-expressing cells were relatively refractory to this effect. Enforced expression of p21WAF1/CJP1 was sufficient to confer nuclear localization on Cdc2 in the p53 null cells, though immunodepletion experiments demonstrated that only a small proportion of Cdc2 was stably associated with p21WAF1/CJP1 in the p53-expressing cells. We conclude that a p53-dependent pathway can operate after exposure of human cells to ionising radiation to promote G2 arrest accompanied by nuclear translocation rather than inhibitory phosphorylation of Cdc2.

Cell cycle-related variation in subcellular localization of eIF3e/INT6 in human fibroblasts.

The Int-6 gene is a site of mouse mammary tumour virus (MMTV) integration in murine tumours and INT6 protein has been identified independently as a subunit (eIF3e) of the eukaryotic translation initiation factor eIF3. In addition, the protein can interact with two other multi-subunit complexes: the COP9 signalosome (CSN) and the proteasome. The role of INT6 in tumourigenesis is nonetheless currently unclear. Here, using immunofluorescence microscopy, we show that eIF3e/INT6 is localized in part to the nucleus, while other eIF3 components are cytoplasmic. Primary human fibroblasts, but not their transformed counterparts, showed reduced nuclear INT6 staining in some cells, and this reduction was maximal in early S phase. This variation in eIF3e/INT6 may indicate regulated shuttling between cellular compartments and would be consistent with the presence of a nuclear export signal as well as a nuclear localization signal in the protein sequence. Loss of regulation of eIF3e/INT6 redistribution may therefore be a significant feature of malignancy in human cells.

Subcellular localisation of cyclin B, Cdc2 and p21(WAF1/CIP1) in breast cancer. association with prognosis.

The heterodimeric cyclin B/Cdc2 protein kinase governs entry into mitosis, and can be negatively regulated through p53-mediated transcriptional induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). Ectopic expression of p21(WAF1/CIP1) in cultured cells has been shown previously to influence the subcellular distribution of the cyclin-dependent kinases (CDKs) including Cdc2. In this study, we have examined the subcellular localisation of Cdc2, cyclin B and p21(WAF1/CIP1) by immunohistochemistry in a well characterised series of primary breast cancers. Surprisingly, p21(WAF1/CIP1) was predominantly cytoplasmic in many of the tumours, where it was associated with high p53 levels; cytoplasmic p21(WAF1/CIP1) and high cyclin B levels were also significant predictors of poor prognosis. We conclude that breast tumorigenesis may be characterised by abnormalities in pathways determining not only levels of expression of key regulatory molecules, but also their subcellular localisation. Investigation of the subcellular distribution of cell cycle regulatory proteins, particularly p21(WAF1/CIP1), could provide valuable prognostic markers in breast cancer.

An oncogenic role of eIF3e/INT6 in human breast cancer.

Altered expression of the eukaryotic translation initiation factor 3 (eIF3) subunit eIF3e/INT6 has been described in various types of human cancer, but the nature of its involvement in tumorigenesis is not yet clear. Using immunohistochemical analysis of 81 primary breast cancers, we found that high tumor grade correlated significantly with elevated cytoplasmic eIF3e level in epithelial tumor cells. Analysis of protein synthesis after siRNA-mediated knockdown in breast cancer cell lines indicated that eIF3e is not required for bulk translation. Microarray analysis of total and polysomal RNAs nonetheless identified distinct sets of mRNAs regulated either positively or negatively by eIF3e; functional classification of these revealed a marked enrichment of genes involved in cell proliferation, invasion and apoptosis. Validated mRNA targets regulated positively at the translational level by eIF3e included urokinase-type plasminogen activator and apoptotic regulator BCL-XL, whereas synthesis of proteins including the mitotic checkpoint component MAD2L1 was negatively regulated. Finally, eIF3e-depleted breast carcinoma cells showed reduced in vitro invasion and proliferation. Taken together, our study data suggest that eIF3e has a positive role in breast cancer progression. It regulates the translation, and in some cases abundance, of mRNAs involved in key aspects of cancer cell biology.

Deregulated gene expression pathways in myelodysplastic syndrome hematopoietic stem cells.

To gain insight into the molecular pathogenesis of the myelodysplastic syndromes (MDS), we performed global gene expression profiling and pathway analysis on the hematopoietic stem cells (HSC) of 183 MDS patients as compared with the HSC of 17 healthy controls. The most significantly deregulated pathways in MDS include interferon signaling, thrombopoietin signaling and the Wnt pathways. Among the most significantly deregulated gene pathways in early MDS are immunodeficiency, apoptosis and chemokine signaling, whereas advanced MDS is characterized by deregulation of DNA damage response and checkpoint pathways. We have identified distinct gene expression profiles and deregulated gene pathways in patients with del(5q), trisomy 8 or -7/del(7q). Patients with trisomy 8 are characterized by deregulation of pathways involved in the immune response, patients with -7/del(7q) by pathways involved in cell survival, whereas patients with del(5q) show deregulation of integrin signaling and cell cycle regulation pathways. This is the first study to determine deregulated gene pathways and ontology groups in the HSC of a large group of MDS patients. The deregulated pathways identified are likely to be critical to the MDS HSC phenotype and give new insights into the molecular pathogenesis of this disorder, thereby providing new targets for therapeutic intervention.

DNA analysis: what and when to request?

Over the last 15 years genetic testing by DNA analysis has expanded enormously both in volume and range due to advances in scientific knowledge and analytical technology. This type of analysis has the potential to provide rapid, cost effective, and accurate diagnostic information but also has its limitations. Some of the changes detected may be of ambiguous consequence and as the knowledge base expands so too does the recognition that other factors can influence the clinical picture. In many cases outcomes may be predicted only on a statistical basis rather than individually. Careful attention should therefore be given to the clinical question that is being addressed before such testing is requested.

Polyomavirus early region alternative poly(A) site: 3'-end heterogeneity and altered splicing pattern.

The position of an alternative polyadenylation [poly(A)] site at the 3' end of the polyomavirus middle T antigen (T-Ag) coding sequences suggests the possibility of a functional role for this site in early gene regulation. The fine structure of this alternative poly(A) site was determined by cDNA sequence and 3' S1 analyses. Cleavage-poly(A) was found to be heterogeneous, occurring at multiple CA dinucleotides downstream from the AATAAA signal sequence. About 50% of the alternative poly(A) takes place upstream from the middle T-Ag stop codon. In addition, the pattern of splicing of transcripts with the alternative poly(A) site differed from that with the major poly(A) site at the end of the early region. The ratio of the small and middle T-Ag splices to the large T-Ag splice for the alternative poly(A)+ mRNAs is about 2.5 times that found for mRNAs with the major poly(A) site. The altered splicing pattern and 3'-end heterogeneity of the alternative poly(A)+ mRNAs would result in preferential translation of small T-Ag (to a greater degree) and middle T-Ag over large T-Ag at later times in the polyomavirus lytic cycle.

Cellular responses to DNA damage.

Cells are constantly under threat from the cytotoxic and mutagenic effects of DNA damaging agents. These agents can either be exogenous or formed within cells. Environmental DNA-damaging agents include UV light and ionizing radiation, as well as a variety of chemicals encountered in foodstuffs, or as air- and water-borne agents. Endogenous damaging agents include methylating species and the reactive oxygen species that arise during respiration. Although diverse responses are elicited in cells following DNA damage, this review focuses on three aspects: DNA repair mechanisms, cell cycle checkpoints, and apoptosis. Because the areas of nucleotide excision repair and mismatch repair have been covered extensively in recent reviews, we restrict our coverage of the DNA repair field to base excision repair and DNA double-strand break repair.

Translation initiation and its deregulation during tumorigenesis.

Regulation of protein synthesis at the level of translation initiation is fundamentally important for the control of cell proliferation under normal physiological conditions. Conversely, misregulation of protein synthesis is emerging as a major contributory factor in cancer development. Most bulk protein synthesis is initiated via recognition of the mRNA 5' cap and subsequent recognition of the initiator AUG codon by a directional scanning mechanism. However, several key regulators of tumour development are translated by a cap-independent pathway. Here we review eukaryotic translation initiation, its regulation and the ways in which this regulation can break down during tumorigenesis.

Regulated expression and phosphorylation of a possible mammalian cell-cycle control protein.

A novel approach to the study of the control of the mammalian cell cycle was opened by the cloning of a human gene by complementation of a fission-yeast cdc2 cell-cycle mutant. We have investigated the behaviour of the RNA and protein products of this human gene, CDC2Hs, and its murine equivalent, CDC2Mm during serum starvation and re-feeding of cultured fibroblasts. In contrast to the pattern of wild-type cdc2+ expression in fission yeast previously described, the mammalian homologue displays variation in both RNA and protein levels during exit from and re-entry into the mitotic cycle. Like its yeast counterpart, however, the mammalian CDC2 protein (p34CDC2) becomes dephosphorylated upon shifting from exponential growth to quiescence, and rephosphorylated late in the G1 phase when cells are stimulated to re-enter the cycle. We propose that phosphorylation of p34CDC2 serves as a regulatory mechanism generally in eukaryotic cells, while transcriptional control of the CDC2 gene in higher eukaryotes may be relevant to long term processes such as senescence and differentiation.

Sequence of Crm1/exportin 1 mutant alleles reveals critical sites associated with multidrug resistance.

We have previously shown that genes involved in a novel pathway of multidrug resistance (MDR) in the fission yeast Schizosaccharomyces pombe are functionally conserved in human cells (V. Spataro et al. (1997) J Biol Chem 272: 30470-30475). The human homologue of one of these genes, hCRM1, has recently been identified and found to function in nucleocytoplasmic export, a process which controls the subcellular localization and hence activity of a number of key cell cycle regulators and transcription factors. Several mutant alleles of crm1 confer a phenotype of MDR in S. pombe, through the nuclear accumulation of the AP-1 transcription factor Pap1. We therefore sequenced mutations of crm1 in fission yeast in order to guide the search for analogous hCRM1 mutations which could play a role in tumour-drug resistance. Fifteen yeast crm1 mutants were assessed by PCR and DNA sequencing. Four mis-sense mutations were identified in the open reading frame, three of which (G to A transitions at nucleotide positions 385, 895 and 1,288) were capable of conferring the MDR phenotype alone. For three of the four mutations found, the corresponding amino acid changes affect residues which are conserved in the human homologue hCRM1 and lie in highly conserved regions of the CRM1 protein. We analysed the corresponding hCRM1 coding regions by RT-PCR and sequencing in a panel of ten tumour cell lines, including three ovarian lines resistant either to cisplatin or paclitaxel, or to both and one MDR breast cancer cell line with nuclear accumulation of the transcription factor YB-1. No hCRM1 mutations were found in the three cDNA fragments examined in this panel of tumour cell lines. However, the identification of amino acid residues within the CRM1 protein that are critical for the export of the MDR-associated transcription factor Pap1 in fission yeast can guide further analysis of hCRM1 mutations in tumours with a MDR phenotype.

The FT210 cell line is a mouse G2 phase mutant with a temperature-sensitive CDC2 gene product.

The mouse cell FT210 was isolated as a G2 phase mutant with a possible defect in the histone H1 kinase. We determined that a temperature-sensitive lesion in this cell line lies in the CDC2 gene. DNA sequence analysis revealed two point mutations in highly conserved regions of the gene: an isoleucine to valine change in the PSTAIR region, and a proline to serine change at the C-terminal region of the protein p34. These mutations cause the p34 protein kinase to become inactivated and degraded in FT210 cells at the restrictive temperature, 39 degrees C. The consequence of this temperature-induced inactivation of the CDC2 gene product is cell cycle arrest at the mid to late G2 phase, and this arrest can be alleviated by the introduction of the human CDC2 homolog.

Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae.

We have characterised two restriction fragments, isolated from a "shotgun" collection of human DNA, which function as autonomously replicating sequences (ARSs) in Saccharomyces cerevisiae. Functional domains of these fragments have been defined by subcloning and exonuclease (BAL 31) deletion analysis. Both fragments contain two spatially distinct domains. One is essential for high frequency transformation and is termed the Replication Sequence (RS) domain, the other, termed the Replication Enhancer (RE) domain, has no inherent replication competence but is essential for ensuring maximum function of the RS domain. The nucleotide sequence of these domains reveals several conserved sequences one of which is strikingly similar to the yeast ARS consensus sequence.

Apoptosis and cytokine release induced by ionizing or ultraviolet B radiation in primary and immortalized human keratinocytes.

We have compared the induction of apoptosis and cytokine release by UVB and gamma-radiation in primary (untransformed) and in two immortalized human epithelial/keratinocyte cell lines, HaCaT and KB (KB is now known to be a subline of the ubiquitous keratin-forming tumour cell line HeLa and we therefore designate it HeLa-KB). In both the primary and the immortalized cell lines apoptosis and release of the inflammatory cytokine interleukin-6 are induced rapidly following UVB irradiation. In contrast, only the immortalized cells undergo apoptosis and release interleukin-6 after gamma-irradiation and here the onset of apoptosis and cytokine release are delayed. The same distinction between primary and immortalized cells was observed when double-strand breaks were induced with the anticancer drug mitoxantrone, which stabilizes topoisomerase II-cleavable complexes. We suggest that immortalization may sensitize keratinocytes to the apoptogenic effect of ionizing radiation or mitoxantrone by deregulating normal cell cycle checkpoints. In both human keratinocytes and fibroblasts, cell killing, as assayed by loss of colony-forming ability, is not coupled to apoptosis. Immortalization increases resistance to gamma-radiation killing but sensitizes to apoptosis. In contrast, although immortalization also sensitizes to UVB-induced apoptosis, it does not affect UVB-induced cell killing. Apoptosis unambiguously indicates death at the single cell level but clonal cell survival integrates all the cellular and genetic processes which prevent or permit a scorable clone to develop.