Adayabalam S. Balajee

RECQL, a member of the RecQ family of DNA helicases, suppresses chromosomal instability

ABSTRACT The mouse gene Recql is a member of the RecQ subfamily of DEx-H-containing DNA helicases. Five members of this family have been identified in both humans and mice, and mutations in three of these, BLM, WRN, and RECQL4, are associated with human diseases and a cellular phenotype that includes genomic instability. To date, no human disease has been associated with mutations in RECQL and no cellular phenotype has been associated with its deficiency. To gain insight into the physiological function of RECQL, we disrupted Recql in mice. RECQL-deficient mice did not exhibit any apparent phenotypic differences compared to wild-type mice. Cytogenetic analyses of embryonic fibroblasts from the RECQL-deficient mice revealed aneuploidy, spontaneous chromosomal breakage, and frequent translocation events. In addition, the RECQL-deficient cells were hypersensitive to ionizing radiation, exhibited an increased load of DNA damage, and displayed elevated spontaneous sister chromatid exchanges. These results provide evidence that RECQL has a unique cellular role in the DNA repair processes required for genomic integrity. Genetic background, functional redundancy, and perhaps other factors may protect the unstressed mouse from the types of abnormalities that might be expected from the severe chromosomal aberrations detected at the cellular level.

Role of the ATPase domain of the Cockayne syndrome group B protein in UV induced apoptosis

Cockayne syndrome (CS) is a human autosomal recessive disorder characterized by many neurological and developmental abnormalities. CS cells are defective in the transcription coupled repair (TCR) pathway that removes DNA damage from the transcribed strand of active genes. The individuals suffering from CS do not generally develop cancer but show increased neurodegeneration. Two genetic complementation groups (CS-A and CS-B) have been identified. The lack of cancer formation in CS may be due to selective elimination of cells containing DNA damage by a suicidal pathway. In this study, we have evaluated the role of the CSB gene in UV induced apoptosis in human and hamster cells. The hamster cell line UV61 carries a mutation in the homolog of the human CSB gene. We show that both human CS-B and hamster UV61 cells display increased apoptotic response following UV exposure compared with normal cells. The increased sensitivity of UV61 cells to apoptosis is complemented by the transfection of the wild type human CSB gene. In order to determine which functional domain of the CSB gene participates in the apoptotic pathway, we constructed stable cell lines with different CSB domain disruptions. UV61 cells were stably transfected with the human CSB cDNA containing a point mutation in the highly conserved glutamic acid residue in ATPase motif II. This cell line (UV61/pc3.1-CSBE646Q) showed the same increased apoptosis as the UV61 cells. In contrast, cells containing a deletion in the acidic domain at the N-terminal end of the CSB protein had no effect on apoptosis. This indicates that the integrity of the ATPase domain of CSB protein is critical for preventing the UV induced apoptotic pathway. In primary human CS-B cells, the induction and stabilization of the p53 protein seems to correlate with their increased apoptotic potential. In contrast, no change in the level of either p53 or activation of mdm2 protein by p53 was observed in hamster UV61 cells after UV exposure. This suggests that the CSB dependent apoptotic pathway can occur independently of the transactivation potential of p53 in hamster cells.

Fine structural analysis of DNA repair in mammalian cells

Nucleotide excision repair (NER) of ultraviolet (UV) light induced photo lesions is heterogeneous in the genomic DNA. We have investigated the mechanistic basis for this repair heterogeneity by analyzing NER activity in higher order chromatin of repair proficient hamster cells. Immunological labeling of repair and transcription sites indicates that NER initiates at the nuclear matrix in close association with transcription. The repair gradually extends into the loop DNA regions in a time dependent fashion. Repair analysis indicates that the DNA damaged by UV irradiation is recruited to the nuclear matrix soon after UV exposure. Consistent with this finding, immunofluorescence and western blotting analyses indicate the enrichment of many NER proteins (XPA, RPA, PCNA, the P62 and p89 sub-units of the basal transcription factor, TFIIH) in the nuclear matrix of UV treated cells. These results strengthen the notion that the nuclear matrix is an important site for the assembly of an efficient repair complex.

Human RecQL4 Helicase Plays Critical Roles in Prostate Carcinogenesis

Prostate cancer is the second leading cause of cancer-associated deaths among men in the western countries. Here, we report that human RecQL4 helicase, which is implicated in the pathogenesis of a subset of cancer-prone Rothmund-Thomson syndrome, is highly elevated in metastatic prostate cancer cell lines. Increased RecQL4 expression was also detected in human prostate tumor tissues as a function of tumor grade with the highest expression level in metastatic tumor samples, suggesting that RecQL4 may be a potential prognostic factor for advanced stage of prostate cancer. Transient and stable suppression of RecQL4 by small interfering RNA and short hairpin RNA vectors drastically reduced the growth and survival of metastatic prostate cancer cells, indicating that RecQL4 is a prosurvival factor for prostate cancer cells. RecQL4 suppression led to increased poly(ADP-ribose) polymerase (PARP) synthesis and RecQL4-suppressed prostate cancer cells underwent an extensive apoptotic death in a PARP-1-dependent manner. Most notably, RecQL4 knockdown in metastatic prostate cancer cells drastically reduced their cell invasiveness in vitro and tumorigenicity in vivo, showing that RecQL4 is essential for prostate cancer promotion. Observation of a direct interaction of retinoblastoma (Rb) and E2F1 proteins with RecQL4 promoter suggests that Rb-E2F1 pathway may regulate RecQL4 expression. Collectively, our study shows that RecQL4 is an essential factor for prostate carcinogenesis.

Werner's syndrome lymphoblastoid cells are hypersensitive to topoisomerase II inhibitors in the G2 phase of the cell cycle.

Werners syndrome (WS) is a rare autosomal recessive human disorder and the patients exhibit many symptoms of accelerated ageing in their early adulthood. The gene (WRN) responsible for WS has been biochemically characterised as a 3-5 helicase and is homologous to a number of RecQ superfamily of helicases. The yeast SGS1 helicase is considered as a human WRN homologue and SGS1 physically interacts with topoisomerases II and III. In view of this, it has been hypothesised that the WRN gene may also interact with topoisomerases II and III. The purpose of this study is to determine whether the loss of function of WRN protein alters the sensitivity of WS cells to agents that block the action of topoisomerase II. This study deals with the comparison of the chromosomal damage induced by the two anti-topoisomerase II drugs, VP-16 and amsacrine, in both G1 and G2 phases of the cell cycle, in lymphoblastoid cells from WS patients and from a healthy donor. Our results show that the WS cell lines are hypersensitive to chromosome damage induced by VP-16 and amsacrine only in the G2 phase of the cell cycle. No difference either in the yield of the induced aberrations or SCEs was found after treatment of cells at G1 stage. These data might suggest that in WS cells, because of the mutation of the WRN protein, the inhibition of topoisomerase II activity results in a higher rate of misrepair, probably due to some compromised G2 phase processes involving the WRN protein.

Efficient PCNA complex formation is dependent upon both transcription coupled repair and genome overall repair.

The protein proliferating cell nuclear antigen (PCNA) is an auxiliary factor for DNA polymerase delta and is involved in the resynthesis step of nucleotide excision repair (NER). After UV irradiation of quiescent cells, PCNA forms an insoluble complex with nuclear substructures. We have investigated associations between NER and its subcomponent pathway, transcription coupled repair (TCR) on PCNA complex formation using genetically related hamster cell lines with different repair characteristics. In DNA repair proficient cells, the PCNA complex was readily detectable within 30 min after UV irradiation by both immunofluorescence and western blot analyses. This complex formation after UV occurs efficiently in quiescent cells. In UV5 (human XP-D homolog) and UV 24 (human XP-B homolog) cells, which are totally deficient in NER, the PCNA complex was not detectable at 30 min after UV. The PCNA complex formation is restored to normal levels in UV5 cells after transfection with the human XPD gene, encoding a subunit of the basal transcription factor, TFIIH. In UV61 (Human CS-B homolog) cells, that are defective only in transcription coupled repair (TCR) of cyclobutane pyrimidine dimers (CPDs), the rate of PCNA complex formation was 2-fold slower than in repair proficient cells. This defect was complemented by transfection of the CSB gene into the UV61 cells. We thus conclude that efficient PCNA complex formation after UV is dependent upon both the NER and TCR pathways in hamster cells. The association of several other DNA repair proteins including XPA, RPA, TFIIH and p53 with the insoluble PCNA complex in UV treated cells suggests a central role for PCNA in different steps of NER.

DNA-dependent protein kinase (DNA-PK)-deficient human glioblastoma cells are preferentially sensitized by Zebularine

Brain tumor cells respond poorly to radiotherapy and chemotherapy due to inherently efficient anti-apoptotic and DNA repair mechanisms. This necessitates the development of new strategies for brain cancer therapy. Here, we report that the DNA-demethylating agent Zebularine preferentially sensitizes the killing of human glioblastomas deficient in DNA-dependent protein kinase (DNA-PK). In contrast to DNA-PK-proficient human glioblastoma cells (MO59K), cytotoxicity assay with increasing Zebularine concentrations up to 300 microM resulted in a specific elevation of cell killing in DNA-PK-deficient MO59J cells. Further, an elevated frequency of polyploid cells observed in MO59J cells after Zebularine treatment pointed out a deficiency in mitotic checkpoint control. Existence of mitotic checkpoint deficiency in MO59J cells was confirmed by the abnormal centrosome number observed in Zebularine-treated MO59J cells. Although depletion of DNA methyltransferase 1 by Zebularine occurred at similar levels in both cell lines, MO59J cells displayed increased extent of DNA demethylation detected both at the gene promoter-specific level and at the genome overall level. Consistent with increased sensitivity, deoxy-Zebularine adduct level in the genomic DNA was 3- to 6-fold higher in MO59J than in MO59K cells. Elevated micronuclei frequency observed after Zebularine treatment in MO59J cells indicates the impairment of DNA repair response in MO59J cells. Collectively, our study suggests that DNA-PK is the major determining factor for cellular response to Zebularine.

Effect of curcumin on irradiated and estrogen-transformed human breast cell lines.

Curcumin (diferuloyl methane) is a well known antioxidant that exerts antiproliferative and apoptotic effects. Curcumin effect was evaluated in a breast cancer model that was developed using the immortalized breast epithelial cell line MCF-10F after exposure to low doses of high LET (linear energy transfer) α particles (150 keV/µm) of radiation, and subsequently cultured in the presence of 17β-estradiol (estrogen). This model consisted of human breast epithelial cells in different stages of transformation: i) MCF-10F; ii) Estrogen cell line; iii) a malignant Alpha3 cell line; iv) a malignant and tumorigenic, Alpha5 cell line; and v) a cell line derived from Alpha5 injected into the nude mice that gave rise to Tumor2 cell line. Curcumin decreased anchorage-independent growh in transformed breast cancer cell lines in comparison to their counterparts and increased the percentage of cells from G₀/G₁ with a concomitant increase in G₂/M phases, as well as a decrease in PCNA and Rho-A protein expression. Among the oncogenes, c-Ha-Ras and Ras homologous A (Rho-A) are important cell signaling factors for malignant transformation and to reach their active GTP bound state, Ras proteins must first release bound GDP mediated by a guanine nucleotide releasing factor (GRF). Then curcumin decrease RasGRF1 protein expression in malignant cell lines. Further, differential expression levels of cleaved (ADP) ribose polymerase 1 (PARP-1) and phosphorylated histone H2AX (γ-H2AX) were observed after curcumin treatment. It seems that PARP-1 similar to H2AX, confers cellular protection against radiation and estrogen-induced DNA damage mediated by curcumin. Therefore, targeting either PARP-1 or H2AX may provide an effective way of maximizing the therapeutic value of antioxidants for cancer prevention.

Protective role of curcumin in oxidative stress of breast cells

Curcumin (diferuloylmethane) is a well known antioxidant that exerts anti-proliferative and apoptotic effects. The effects of curcumin were evaluated in a breast cancer model that was developed with the immortalized breast epithelial cell line, MCF-10F after exposure to low doses of high LET (linear energy transfer) α particles (150xa0keV/µm) of radiation, and subsequently cultured in the presence of 17β-estradiol (estrogen). This model consisted of human breast epithelial cells in different stages of transformation: i) a control cell line, MCF-10F, ii) an estrogen-treated cell line, named Estrogen, iii) a malignant cell line, named Alpha3 and iv) a malignant and tumorigenic, cell line named Alpha5. Curcumin decreased the formation of hydrogen peroxide in the control MCF-10F, Estrogen and Alpha5 cell lines in comparison to their counterparts. Curcumin had little effect on NFκB (50xa0kDa) but decreased the protein expression in the Estrogen cell line in comparison to their counterparts. Curcumin enhanced manganese superoxide dismutase (MnSOD) protein expression in the MCF-10F and Alpha3 cell lines. Results indicated that catalase protein expression increased in curcumin treated-Alpha3 and Alpha5 cell lines. Curcumin slightly decreased lipid peroxidation in the MCF-10F cell lines, but significantly (P<0.05) decreased it in the Alpha5 cell line treated with curcumin in comparison to their counterparts as demonstrated by the 8-iso-prostaglandin F2α (8-iso-PGF2α) levels. It can be concluded that curcumin acted upon oxidative stress in human breast epithelial cells transformed by the effect of radiation in the presence of estrogen.