Upregulation of RAD51 expression is associated with progression of thyroid carcinoma.

AIMS: RAD51 participates in homologous recombination repair (HRR) of double-stranded DNA breaks (DSBs) which may cause genomic instability and cancer. The aim of this study was to investigate RAD51 gene expression at transcriptional and translational levels to measure mRNA and protein level and to correlate its relationship with proliferation marker, Ki67 in thyroid cancer patients. This study also explored correlation of these genes with different clinicopathological parameters of the study cohort by Spearman's rank correlation coefficient. METHODS: Quantitative real time polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to detect mRNA transcript levels and protein expression of RAD51 and Ki67 in 102 cases of thyroid cancer tissues and equal number of uninvolved healthy thyroid tissue controls. RESULTS: Data showed that expression for both RAD51 and Ki67 was significantly increased in thyroid cancer (p<0.001). High RAD51 and Ki67 expression was associated with later stages, poor tissue differentiation, large tumor size, positive lymph node metastasis and distant metastasis. The correlation analysis demonstrated a strong positive correlation (r=0.461) between RAD51 and Ki67 on mRNA level and on protein level (r=0.866). Strong correlation was observed between clinicopathological characteristics and selected molecules. CONCLUSION: The present study concluded that upregulation of RAD51 and overexpression of Ki67 may be associated with the progression of thyroid cancer.

The detection and assessment of the aneugenic potential of selected oestrogens, progestins and androgens using the in vitro cytokinesis blocked micronucleus assay.

The use of 17-beta-oestradiol, testosterone, progesterone, zearanol, trenbolone acetate and melengesterol acetate in animal feed as growth promoters has been banned in the European Union since 1989. However, the data available on their genotoxicity is limited. To bridge this gap the present study was carried out with the aim of evaluating these hormones for their ability to induce aneuploidy. Aneuploidy has been recently considered sufficiently important to be included in the routine testing of chemicals and radiation. These types of numerical chromosomal aberrations may arise by at least two mechanisms, chromosome loss and non-disjunction. Over the past few years, the cytokinesis blocked micronucleus (CBMN) technique has evolved into a robust assay for the detection of aneuploidy induction. At the present time, it is the only assay which can reliably detect both chromosome loss and non-disjunction when the basic methodology is coupled with appropriate molecular probing techniques such as immunoflourescent labelling of kinetochores and Fluorescence in situ Hybridisation. In this present study, aneuploidy induction by three groups of hormones was studied using CBMN assay coupled with Fluorescence in situ Hybridisation. The results from the present study demonstrate that 17-beta-oestradiol, diethylstilboestrol, progesterone and testosterone are genotoxic and induce aneuploidy by non-disjunctional mechanism, whereas trenbolone is also genotoxic by a clastogenic mechanism. However, melengesterol acetate and zearanol proved to be non-genotoxic in vitro.

Novel SYK gene variations and changes in binding sites of miRs in breast cancer patients.

BACKGROUND: Spleen Tyrosine Kinase (SYK) belongs to non-receptor tyrosine Kinase family, which normally expresses in epithelial breast tissues and acts as a tumor suppressor gene. OBJECTIVE: Analysis of mutations in the SYK gene and deregulation of SYK transcripts by miRNA in breast cancer was studied. METHODS: All exons and exon/intron boundaries of SYK gene were amplified and sequenced in blood samples of 207 breast cancer cases and 200 matched controls using PCR-single stranded conformational polymorphism method. RESULTS: Sequence analysis revealed 10 novel mutations in breast cancer patients. Among these 6 mutations (Ala 161Pro, His162Tyr, Phe191Tyr, Val 535Gly, Ser 556lIe and Lys536Gln) were found in exonic region and 4 (26249 T>A, 63941 G>A, 63981G>C and 86548T>A) were found in intronic region. All of these mutations are associated with ∼ 5 folds (p< 0.0001) increase in breast cancer risk in present study cohort. Regulation of SYK transcripts by miRNA was also analyzed using in silico bioinformatics tools, exon 6's mutation (Phe191Tyr) was found to have altered interaction with miR-873. CONCLUSION: Overall novel mutations in SYK gene and in silico analysis revealed that these mutations are crucial and might be responsible for altered expression of SYK.

Acetylation of retinoblastoma like protein2 (Rb2/p130) in tumor tissues.

The activity of Rb proteins is controlled by post-translational modifications, especially through phosphorylation. Acetylation of Rb2/p130 was reported recently in NIH3T3 cells but its physiological relevance in cell cycle control and tumorigenesis is still unknown. Efforts are underway to investigate possible interplay between Rb2/p130 phosphorylation and acetylation. Here we hypothesized that Rb2/p130 acetylation, like p53 acetylation, may play a role in development of the tumor phenotype. The proposed hypothesis regarding acetylation of Rb2/p130 in tumor VS normal cells was found to be true in our case study of 36 tumor samples. Statistical analysis of results suggest strong correlation among Rb2/p130 acetylation and cancer phenotype.

Lack of influence of glutathione S-transferase gene deletions in sporadic breast cancer in Pakistan.

Glutathione S-transferases constitute the phase II detoxification enzymes involved in the metabolism and detoxification of a wide range of potential environmental carcinogens. GSTM1 and GSTT1 are polymorphic and their deletions have been found to be associated with breast cancer risk in some of the world populations. The current study was aimed at evaluation of GSTM1 and GSTT1 gene deletions in 150 unrelated breast cancer patients and 150 healthy controls from Pakistani population. Multiplex PCR assay along with CYP1A1 exon 7 as an internal control was used. Our sampled patients and controls had a mean age of 48 (+11.8) and 45 (+7.9) years respectively. The analysis suggested that only 2% breast cancer patient and 8% controls had homozygous GSTM1 gene deletions (OR 0.23, 95% CI 0.06-0.85). A total of 8.7% patients and 18.6% controls had homozygous GSTT1 deletion (OR 0.41, 95% CI 0.25-0.83). The statistical analysis suggest that a non significant number (P>0.05) of individuals compared to controls have GSTM1 and GSTT1 gene deletions. Deletion in both genes was not observed in any of the patients or controls. The present case control study suggests no association of GSTM1 and GSTT1 gene deletions with sporadic form of breast cancer in Pakistani population.

Expressional alterations and transcript isoforms of metastasis suppressor genes (KAI1 and KiSS1) in breast cancer patients.

BACKGROUND: Metastasis suppressor genes are involved in the inhibition of a cancer cell's ability to metastasize. Down expression of such genes may contribute to pathogenesis of breast cancer. The aim of current study was firstly to evaluate expression of two examples, KAI1 and KISS1, and then to determine relationships with stages of breast cancer in a Pakistani population. METHODOLOGY: Fresh biopsy tissues were collected from different hospitals and oncology research institutes. The semi quantitative reverse transcriptase polymerase chain reaction was used to investigate KAI1 and KISS1 expression in 25 breast tumor tissues and 25 normal tissues. Statistical analysis was performed to explore its association with breast cancer risk. RESULTS: The present study revealed that KAI1 and KISS1 mRNA expression was markedly reduced in tissues of breast cancer compared to adjacent normal tissue. In present study a splice variant of KAI1 during a screen for its expression analysis was also observed. This splice variant has not been reported previously. CONCLUSIONS: Metastasis suppressor genes demonstrate reduced expression in breast cancers in Pakistan.

The in vitro genotoxicity of ethanol and acetaldehyde.

Ability of ethanol to produce chromosomal changes has been controversial in past many years; nevertheless many recent studies have shown that ethanol itself produces genotoxic effects like acetaldehyde. This study was carried out to evaluate the ability of ethanol and its metabolite acetaldehyde to induce chromosomal changes using in vitro CBMN assay (Cytokinesis Blocked Micronucleus assay) in conjunction with immunofluorescent labeling of kinetochores. Kinetochore staining was used with a view to differentiate, between the genotoxic effects of both chemicals, and ascertain the mechanisms of genotoxicity induction by ethanol and acetaldehyde. Both ethanol and acetaldehyde produced statistically significant (P<0.05) dose dependent increase in MN induction as compared with the controls over the dose range tested. Kinetochore analysis proved that the MN induced in ethanol were originated by an aneugenic mechanism, whereas in the case of acetaldehyde most of the MN had originated by a clastogenic mechanism. This not only confirms the ability of ethanol to produce DNA damage in vitro but it also establishes the efficacy of CBMN assay to detect and differentiate between the genotoxic effects of different genotoxins. Here we report that ethanol is itself genotoxic, at least in vitro, and produces genotoxic effects mainly through an aneugenic mechanism whereas its metabolite acetaldehyde is a clastogen.

Association of GSTM1 and GSTT1 gene deletions with risk of head and neck cancer in Pakistan: a case control study.

Polymorphic deletions of GSTM1 and GSTT1 genes involved in the detoxification of potentially carcinogenic agents may be risk factors for various cancers, including head and neck cancer (HNC). In the present case-control study we aimed to access possible associations of HNC with GSTM1 and GSTT1 null genotypes in a Pakistani population. DNA was extracted from leukocytes of 388 cancer patients and 150 healthy controls by phenol-chloroform procedure. GSTM1 and GSTT1 deletion variants were genotyped by multiplex PCR assay with CYP1A1 as an internal control and further analyzed by primer specific PCR assay and sequencing. Mean age of cases and controls was 48 (±16.6) years with a male to female ratio of 1:1. Cancer of the oral cavity (57%) was most prevalent in the sampled population followed by pharynx and larynx (30% and 13% respectively). A statistically significant (P<0.05) association was observed for both null genotypes in contribution to HNC as compared with the controls. The odds ratio (OR) for the GSTM1 null genotype was 2.3 with a 95% CI of 1.5-5.5 and for GSTT1 OR was 2.04 with 95% CI of 1.3-3.1. These results suggest that the GSTM1 and GSTT1 null genotypes are risk factors for HNC development among the Pakistani population.