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PubMed2004

Global gene expression profile of nasopharyngeal carcinoma by laser capture microdissection and complementary DNA microarrays.

A number of genetic and epigenetic changes underlying the development of nasopharyngeal carcinomas have recently been identified. However, there is still limited information on the nature of the genes and gene products whose aberrant expression and activity promote the malignant conversion of nasopharyngeal epithelium. Here, we have performed a genome-wide transcriptome analysis by probing cDNA microarrays with fluorescent-labeled amplified RNA derived from laser capture microdissected cells procured from normal nasopharyngeal epithelium and areas of metaplasia-dysplasia and carcinoma from EBV-associated nasopharyngeal carcinomas. This approach enabled the identification of genes differentially expressed in each cell population, as well as numerous genes whose expression can help explain the aggressive clinical nature of this tumor type. For example, genes indicating cell cycle aberrations (cyclin D2, cyclin B1, activator of S-phase kinase, and the cell cycle checkpoint kinase, CHK1) and invasive-metastatic potential (matrix metalloproteinase 11, v-Ral, and integrin beta(4)) were highly expressed in tumor cells. In contrast, genes underexpressed in tumors included genes involved in apoptosis (B-cell CLL/lymphoma 6, secretory leukocyte protease inhibitor, and calpastatin), cell structure (keratin 7 and carcinoembryonic antigen-related cell adhesion molecule 6), and putative tumor suppressor genes (H-Ras-like suppressor 3, retinoic acid receptor responder 1, and growth arrested specific 8) among others. Gene expression patterns also suggested alterations in the Wnt/beta-catenin and transforming growth factor beta pathways in nasopharyngeal carcinoma. Thus, expression profiles indicate that aberrant expression of growth, survival, and invasion-promoting genes may contribute to the molecular pathogenesis of nasopharyngeal carcinoma. Ultimately, this approach may facilitate the identification of clinical useful markers of disease progression and novel potential therapeutic targets for nasopharyngeal carcinoma.

Carcinoma↗
PubMed2004

Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1.

The major regulator of the gap-1/synthesis phase (G(1)/S) cell cycle checkpoint is the retinoblastoma protein (pRb), and this is regulated in part by the activities of cyclin-dependent kinase (cdk)-4 and the D cyclins. Surprisingly, given the potential importance of beta-cell replication for islet replacement therapy, pRb presence, phosphorylation status, and function have not been explored in beta-cells. Here, adenoviruses expressing cdk-4 and cyclin D(1) were used to explore rat and human pRb phosphorylation and beta-cell cycle control. pRb is present in rat and human islets, and overexpression of cyclin D(1)/cdk-4 led to strikingly enhanced pRb phosphorylation in both species. Combined overexpression of both cdk-4 and cyclin D(1) caused a threefold increase in [(3)H]thymidine incorporation. This increase in proliferation was confirmed independently using insulin and bromodeoxyuridine immunohistochemistry, where human beta-cell replication rates were increased 10-fold. Cdk-4 or cyclin D(1) overexpression did not adversely effect beta-cell differentiation or function. The key cell cycle regulatory protein, pRb, can be harnessed to advantage using cyclin D(1)/cdk-4 for the induction of human and rodent beta-cell replication, enhancing replication without adversely affecting function or differentiation. This approach will allow detailed molecular study of the cellular mechanisms regulating the cell cycle in beta-cells, beta-cell lines, and stem cell-derived beta-cells.

Adenoviridae↗
PubMed2005

DNA damage-induced association of ATM with its target proteins requires a protein interaction domain in the N terminus of ATM.

The ATM protein kinase regulates the response of the cell to DNA damage by associating with and then phosphorylating proteins involved in cell cycle checkpoints and DNA repair. Here, we report on deletion studies designed to identify protein domains required for ATM to phosphorylate target proteins and to control cell survival following exposure to ionizing radiation. Deletion studies demonstrated that amino acids 1-150 of ATM were required for the ATM protein to regulate cellular radiosensitivity. Additional deletions and point mutations indicated that this domain extended from amino acids 81-106 of ATM, with amino acid substitutions located between amino acids 91 and 97 inactivating the functional activity of ATM. When ATM with mutations in this region (termed ATM90) was expressed in AT cells, it was unable to restore normal radiosensitivity to the cells. However, ATM90 retained normal kinase activity and was autophosphorylated on serine 1981 following exposure to DNA damage. Furthermore, wild-type ATM displayed DNA-damage induced association with p53, brca1, and LKB1 in vivo, whereas ATM90 failed to form productive complexes with these target proteins either in vivo or in vitro. Furthermore, ATM90 did not phosphorylate p53 in vivo and did not form nuclear foci in response to ionizing radiation. We propose that amino acids 91-97 of ATM contain a protein interaction domain required for the DNA damage-induced association between ATM and its target proteins, including the brca1, p53, and LKB1 proteins. Furthermore, this domain of ATM is required for ATM to form nuclear foci following exposure to ionizing radiation.

AMP-Activated Protein Kinase Kinases↗
PubMed1997

Ataxia-telangiectasia: is ATM a sensor of oxidative damage and stress?

Ataxia-telangiectasia (A-T) is a pleiotropic recessive disorder characterized by cerebellar ataxia, immunodeficiency, specific developmental defects, profound predisposition to cancer and acute radiosensitivity. Functional inactivation of a single gene product, ATM, accounts for this compound phenotype. We suggest that ATM acts as a sensor of reactive oxygen species and/or oxidative damage of cellular macromolecules, including DNA. In turn, ATM induces signalling through multiple pathways, thereby coordinating acute phase stress responses with cell cycle checkpoint control and repair of oxidative damage. Absence of ATM is proposed to limit the repair of insidious oxidative damage that can occur under normal physiological conditions, ultimately leading to apoptosis of particularly sensitive cells, such as neurons and thymocytes.

Apoptosis↗
PubMed1997

P53 and p21 (Waf1/Cip1) mRNA expression associated with DNA damage and repair in acute immune complex alveolitis in mice.

The tumor suppresser p53 is a cell cycle checkpoint protein that contributes to the preservation of genetic stability by mediating either a G1 arrest or apoptosis in response to DNA damage. p53 causes growth arrest through transcriptional activation of the cyclin-dependent kinase inhibitor p21. During p53-mediated suppression of cell proliferation, p21 is important for coordinating cell cycle progression, DNA replication, and repair of damaged DNA. The purpose of this study is to investigate the expression of p53 and p21 mRNA in association with DNA damage and normal repair in acute immune complex alveolitis in mice. Male ICR mice were injected intravenously with IgG antibodies against oval albumin, aerosolized with oval albumin solution, and killed at 4, 6, 12, 24, and 48 hours and 1 week after aerosolization. We assessed the expression of p53 and p21 mRNA by reverse transcriptase (RT)-PCR and by RT in situ PCR. We also assessed DNA damage by terminal deoxynucleotidyl transferase mediated biotin-dUTP nick-end-labeling (TUNEL) and by gel electrophoresis of DNA extracted from lung tissues. The results of RT-PCR and RT in situ PCR showed that p53 and p21 mRNA were concurrently up-regulated at 4 to 48 hours after aerosolization in alveolar epithelial cells. Bronchial and alveolar epithelial cells were positively stained by TUNEL in this period but not at 1 week after aerosolization or in control mice. The result of electrophoretic analysis of DNA was compatible with that of TUNEL. These studies suggest that the responses of p53 and p21 mRNA are associated with physiologic processes of DNA damage and repair in acute immune complex alveolitis in mice.

Animals↗
PubMed1999

Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation.

ATM is a member of the large phosphatidylinositol-3 kinase family and plays an important role in cellular response to DNA damage. To further define the physiological roles of ATM at the cellular level, we created an isogenic set of stable cell lines differing only in their ATM status from the chicken B cell line DT40 by targeted integration. These stable DT40 cell lines, as most of transformed chicken cell lines, do not express p53. However, ATM-/- DT40 cells displayed retarded cellular proliferation, defective G2/M checkpoint control and radio-resistant DNA synthesis. Furthermore, ATM-/- DT40 cells were sensitive to ionizing radiation and showed highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In addition, a slight but significant reduction in targeted integration frequency was observed in ATM-/- DT40 cells. These results suggest that ATM has multiple p53-independent functions in cell cycle checkpoint control and in maintenance of chromosomal DNA. These ATM deficient DT40 clones therefore provide a useful model system for analysing p53-independent ATM functions.

Animals↗
PubMed2004

Living with DNA breaks is an everyday reality for cells adapted to high NaCl.

A rapid, coordinated response to DNA breaks, including activation of cell cycle checkpoints and initiation of accurate DNA repair is believed to be necessary to maintain genomic integrity and prevent accumulation of mutations. That is why it was so unexpected to discover recently that in the mouse renal inner medulla the otherwise healthy cells contain numerous DNA breaks, yet they survive and function adequately. The DNA breaks in the renal inner medulla are caused by the high NaCl concentrations to which the cells are constantly exposed as a consequence of the urinary concentrating mechanism. Cells adapted to high NaCl in cell culture also contain many DNA breaks. The DNA breaks do not trigger cell cycle arrest or cause apoptosis, and the cells safely proliferate rapidly despite their presence. Further, high NaCl inhibits the activity of key components of the classical DNA damage response such as Mre11, chk1 and H2AX. In order to explain why the DNA breaks do not cause disabling mutations, oncogenic transformations and/or apoptosis we speculate that in the presence of high NaCl there might be alternative DNA damage response pathways or special ways of coping with DNA damage.

Animals↗
PubMed2004

The INK4a /ARF locus: role in cell cycle control for renal cell epithelial tumor growth after the Chernobyl accident.

Previous studies have shown that during the period subsequent to the Chernobyl accident, increases in morbidity, aggressivity and proliferative activity of renal-cell carcinomas (RCCs) in Ukrainian patients were recognized. The present paper describes the molecular alterations of those tumor suppressor genes located on chromosome 9p21 ( INK4a/ARF locus and p15(INK4B)) in 26 primary renal-cell epithelial tumors from patients with different degrees of radiation exposure after the Chernobyl accident in Ukraine. Radiometric measurement of Cesium 137 ((137)Cs) was conducted with 1-day urine from all patients before surgery. Our results demonstrate that RCCs from patients living in the radio-contaminated areas showed aberrant hypermethylation of p14(ARF) and p16(INK4A) genes, associated with increased p38MAPK, p14(ARF), mdm2, cyclinD1 and Ki67 protein expression levels. Present findings show the possibility that chronic long-term low-dose radiation activates the INK4a/ARF locus, targeted by activation of the p38MAPK cascade. These actions could lead to disruptions and loss of cell cycle checkpoints and, thereby, to cellular transformation.

Adult↗
PubMed2001

NF-kappaB and cell-cycle regulation: the cyclin connection.

The cyclins are a family of proteins that are centrally involved in cell cycle regulation and which are structurally identified by conserved "cyclin box" regions. They are regulatory subunits of holoenzyme cyclin-dependent kinase (CDK) complexes controlling progression through cell cycle checkpoints by phosphorylating and inactivating target substrates. CDK activity is controlled by cyclin abundance and subcellular location and by the activity of two families of inhibitors, the cyclin-dependent kinase inhibitors (CKI). Many hormones and growth factors influence cell growth through signal transduction pathways that modify the activity of the cyclins. Dysregulated cyclin activity in transformed cells contributes to accelerated cell cycle progression and may arise because of dysregulated activity in pathways that control the abundance of a cyclin or because of loss-of-function mutations in inhibitory proteins.Analysis of transformed cells and cells undergoing mitogen-stimulated growth implicate proteins of the NF-kappaB family in cell cycle regulation, through actions on the CDK/CKI system. The mammalian members of this family are Rel-A (p65), NF-kappaB(1) (p50; p105), NF-kappaB(2) (p52; p100), c-Rel and Rel-B. These proteins are structurally identified by an amino-terminal region of about 300 amino acids, known as the Rel-homology domain. They exist in cytoplasmic complexes with inhibitory proteins of the IkappaB family, and translocate to the nucleus to act as transcription factors when activated. NF-kappaB pathway activation occurs during transformation induced by a number of classical oncogenes, including Bcr/Abl, Ras and Rac, and is necessary for full transforming potential. The avian viral oncogene, v-Rel is an NF-kappaB protein. The best explored link between NF-kappaB activation and cell cycle progression involves cyclin D(1), a cyclin which is expressed relatively early in the cell cycle and which is crucial to commitment to DNA synthesis. This review examines the interactions between NF-kappaB signaling and the CDK/CKI system in cell cycle progression in normal and transformed cells. The growth-promoting actions of NF-kappaB factors are accompanied, in some instances, by inhibition of cellular differentiation and by inhibition of programmed cell death, which involve related response pathways and which contribute to the overall increase in mass of undifferentiated tissue.

Animals↗
PubMedDate not supplied

Checkpoint effectors CDKN1A and Gadd45 correlate with oxidative DNA damage in human prostate carcinoma.

BACKGROUND: Although cellular oxidative stress is a major cause of DNA damage, it is still not clear to what degree it affects genetic instability and malignant progression in established prostate carcinoma (PCa). MATERIALS AND METHODS: We examined the expression of CDKN1A and Gadd45 proteins acting on cell cycle checkpoints and DNA repair in PCa relative to the presence of oxidative DNA damage, as measured by the detection of the DNA adduct 8-hydroxy-2-deoxyguanosine (8-OHdG.). Sixteen PCa and 28 benign prostate hyperplasias (BPH) were analyzed. RT-PCR was used to evaluate WAF1 and Gadd45 transcripts. Western blot and ELISA were used to analyze proteins and 8-OHdG adducts. Proliferation was studied by Ki67 image cytometry; telomerase activity was detected by TRAP- ELISA. RESULTS: Multivariate factor analysis provided evidence that, in PCa, DNA checkpoint proteins were associated with 8-OHdG and did not prevent neoplastic cells proliferation. Conversely, in BPH, oxidative DNA damage was inversely correlated with DNA checkpoint proteins and proliferation, suggesting the presence of energy-depleted senescent cells. CONCLUSION: Although in non-malignant tissue extensive oxidative DNA damage drives cells to a metabolic blockage, in PCa neoplastic cells it activates repair mechanisms favoring the escape from senescence and the expansion of DNA-damaged clones.

Aged↗
PubMed2002

Cell cycle dysregulation by HTLV-I: role of the tax oncoprotein.

HTLV-I is a human retrovirus which is the etiological agent for adult T-cell leukemia. The virus encodes a 40 kDa oncoprotein, Tax, which has no cellular counterpart. Findings from several laboratories over the past decade have shown that over-expression of the Tax oncoprotein is wholly sufficient to transform animal cells. Emerging evidence supports that Tax transforms cells through dysregulation of several cell cycle checkpoints. Here, we review extant data on how Tax targets cyclins, inhibitors of cyclin dependent kinase, as well as cellular sentries for DNA-damage.

Animals↗
PubMed2003

Loss of p53 function accelerates acquisition of telomerase activity in indefinite lifespan human mammary epithelial cell lines.

We describe novel effects of p53 loss on immortal transformation, based upon comparison of immortally transformed human mammary epithelial cell (HMEC) lines lacking functional p53 with closely related p53(+) lines. Our previous studies of p53(+) immortal HMEC lines indicated that overcoming the stringent replicative senescence step associated with critically short telomeres (agonescence), produced indefinite lifespan lines that maintained growth without immediately expressing telomerase activity. These telomerase(-) 'conditionally immortal' HMEC underwent an additional step, termed conversion, to become fully immortal telomerase(+) lines with uniform good growth. The very gradual conversion process was associated with slow heterogeneous growth and high expression of the cyclin-dependent kinase inhibitor p57(Kip2). We now show that p53 suppresses telomerase activity and is necessary for the p57 expression in early passage p53(+) conditionally immortal HMEC lines, and that p53(-/-) lines exhibit telomerase reactivation and attain full immortality much more rapidly. A p53-inhibiting genetic suppressor element introduced into early passages of a conditionally immortal telomerase(-) p53(+) HMEC line led to rapid induction of hTERT mRNA, expression of telomerase activity, loss of p57 expression, and quick attainment of uniform good growth. These studies indicate that derangements in p53 function may impact malignant progression through direct effects on the conversion process, a potentially rate-limiting step in HMEC acquisition of uniform unlimited growth potential. These studies also provide evidence that the function of p53 in suppression of telomerase activity is separable from its cell cycle checkpoint function.

Breast↗
PubMed2006

Postreplication repair and PCNA modification in Schizosaccharomyces pombe.

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays a crucial role in regulating replication past DNA damage in eukaryotes, but the detailed mechanisms appear to vary in different organisms. We have examined the modification of PCNA in Schizosaccharomyces pombe. We find that, in response to UV irradiation, PCNA is mono- and poly-ubiquitinated in a manner similar to that in Saccharomyces cerevisiae. However in undamaged Schizosaccharomyces pombe cells, PCNA is ubiquitinated in S phase, whereas in S. cerevisiae it is sumoylated. Furthermore we find that, unlike in S. cerevisiae, mutants defective in ubiquitination of PCNA are also sensitive to ionizing radiation, and PCNA is ubiquitinated after exposure of cells to ionizing radiation, in a manner similar to the response to UV-irradiation. We show that PCNA modification and cell cycle checkpoints represent two independent signals in response to DNA damage. Finally, we unexpectedly find that PCNA is ubiquitinated in response to DNA damage when cells are arrested in G2.

DNA Damage↗
PubMed2003

A case-control study of cyclin D1 CCND1 870A-->G polymorphism and bladder cancer.

Expression of cyclin D1 is believed to lead to progression through the G1-S cell cycle checkpoint, and both experimental and pathological evidence suggest that over-expression of this protein may increase the risk of several cancers, including transition cell carcinoma of the bladder. Two major transcripts have been described for CCND1, the gene encoding cyclin D1. CCND1 870A-->G, a common single nucleotide polymorphism in the splice donor region of exon 4, may modulate expression of these transcripts, with the A variant resulting in an increased pool of the isozyme encoded by transcript form b. A statistically significant 1.8-fold increased risk for bladder cancer among individuals possessing the A/A genotype was recently reported in a hospital-based case-control study conducted among native Japanese. We conducted a population-based case-control study of incidence of bladder cancer among non-Hispanic whites in Los Angeles County to examine the relationship between CCND1 870A-->G genotypes and bladder cancer risk. No association between the A/A genotype and risk was observed (odds ratio = 0.90, 95% confidence interval 0.60-1.33). The null association was not appreciably modified by bladder cancer risk factors, including lifetime smoking history, or by histopathologic classification.

Adult↗
PubMedDate not supplied

Melanocyte development and malignant melanoma.

Malignant melanoma is a notoriously aggressive disease that can affect relatively young individuals and whose incidence is rising at an alarming rate. Unlike many cancers, metastatic melanoma is poorly responsive to current therapies and mutations affecting p53, the retinoblastoma gene product or Ras which occur frequently in many other cancer types, appear to be rare or at least relatively late events in the progression of the disease. Recent advances in our understanding of the disease at the molecular level have indicated that in addition to the loss of cell cycle checkpoints which may be common to all cancers, malignant melanoma shares many characteristics in common with developmental precursors to melanocytes, the mature pigment producing cells of the skin and hair follicles which are responsible for skin and hair colour. This review therefore focuses on the signalling pathways that play a crucial role in the development of the melanocyte lineage which are subject to deregulation in malignant melanoma namely signalling by receptor tyrosine kinases, the Wnt signalling pathway, as well as loss of the p16INK4a cyclin-dependent kinase inhibitor. Intriguingly all three pathways impact on the expression or function of the microphthalmia-associated transcription factor which plays an essential role in melanocyte development.

Animals↗
PubMed2004

The adaptive imbalance to genotoxic stress: genome guardians rear their ugly heads.

An adaptive response of the genome-protection machinery occurs in cells exposed to genotoxic stress. This machinery includes the p53 and retinoblastoma protein pathways, which are not mutually exclusive from other adapting machinery including DNA repair, cell cycle checkpoints, apoptosis and endogenous metabolizing and antioxidant enzymes. The adaptive changes occur in chronic inflammation and in cigarette smokers associated with a high cancer risk, and are an attempt to keep cells healthy. However, there is increasing evidence that this response may have deleterious effects. Here, key pathways that adaptively respond to genotoxic stress are reviewed and mechanisms by which this response may have pro-carcinogenic implications are discussed.

Animals↗
PubMed2006

Heat-induced phosphorylation of NBS1 in human skin fibroblast cells.

NBS1 is known to be involved in DNA damage-induced cellular responses after exposure to ionizing radiation (IR). Phosphorylation of NBS1 contributes to cell-cycle checkpoints. The aim of this study was to determine whether heat exposure induces or stimulates cellular responses mediated by the phosphorylation of NBS1 in human skin fibroblast cell lines. The results of immunofluorescent staining and Western blot analysis showed that NBS1 proteins are phosphorylated after exposure to heat in the nucleus of a normal skin fibroblast cell line (82-6 cells). This suggests that the NBS1-mediated signal transduction could be induced by heat. We further examined whether a deficiency in the NBS1 protein modifies heat sensitivity in human skin fibroblast cell lines. A skin fibroblast cell line (Gmtert), derived from a Nijmegen breakage syndrome (NBS) patient containing mutant NBS1, showed higher sensitivity to heat than the same cell line transfected with the wild-type copy of the NBS1 gene. We also showed that transfection of a DNA cassette expressing small interference RNA (siRNA) targeted to NBS1 into 82-6 cells enhanced cell sensitivity to heat. These results suggest that NBS1 is involved in cellular responses to DNA damage which is induced by heat exposure as well as by radiation exposure in human skin fibroblast cells.

Base Sequence↗
PubMed2003

Genomic instability and endoreduplication triggered by RAD17 deletion.

Cell cycle checkpoints are critical for genomic stability. Rad17, a component of the checkpoint clamp loader complex (Rad17/Rfc2-5), is required for the response to DNA damage and replication stress. To explore the role of Rad17 in the maintenance of genomic integrity, we established somatic conditional alleles of RAD17 in human cells. We find that RAD17 is not only important for the Atr-mediated checkpoint but is also essential for cell viability. Cells lacking RAD17 exhibited acute chromosomal aberrations and underwent endoreduplication at a high rate. Therefore, RAD17 links the checkpoint to ploidy control and is essential for the maintenance of chromosomal stability.

Blotting, Southern↗