Mohammed Kunhi

Cellular Radiosensitivity, Radioresistant DNA Synthesis, and Defect in Radioinduction of p53 in Fibroblasts From Atherosclerosis Patients

Earlier studies have suggested that both cancer and atherosclerosis may follow a common pathway in the early stage of development and share certain risk factors. One report indicated that the gene responsible for the radiosensitive, cancer-prone, multisystem disorder ataxia telangiectasia (AT) may increase the risk of developing ischemic heart disease. The present studies were carried out to find similarities, if any, between atherosclerosis patients and AT homozygotes or heterozygotes (ATHs) in their cellular/molecular response to ionizing radiation, which acts as a carcinogen as well as an atherogen. Fibroblast cell strains developed from healthy subjects and from AT homozygotes, ATHs, and atherosclerosis patients were compared for (1) survival, by the colony-forming assay and (2) DNA synthesis inhibition after irradiation, determined by [3H]thymidine incorporation, cell cycle distribution, and the expression of p53 and p21 proteins, analyzed by flow cytometry. Fibroblasts from the atherosclerosis patients as a group, compared with the healthy subjects, showed enhanced sensitivity to chronic (low-dose-rate) irradiation. A majority of the cell strains representing atherosclerosis patients exhibited varying degrees of radioresistant DNA synthesis (RDS), with roughly 33% showing an AT-like and the rest an ATH-like response. All cell strains with an AT-like and one quarter with an ATH-like RDS were found to be defective in the radioinduction of both p53 and p21 proteins, which are concerned with cell cycle regulation. An absence of G1 arrest after irradiation was observed in cell strains lacking a radioinduced expression of p53 and p21. Cellular/molecular defects leading to increased radiosensitivity, reduced induction of p53/p21, and cell cycle deregulation found to be associated with cancer-prone disorders such as AT may constitute important risk factors for atherosclerosis as well.

ROLE OF INTERMEDIARY BIOMARKERS IN DETERMINING THE ANTICANCER EFFICACY OF MARINE COMPOUNDS

In the present study, two of the probable an umor marine compounds, manzamine A and sarcophine, were screened using benzo[a]pyrene (BP)-derived DNA adduct formation in MCF-7 cells as intermediary biomarker. Briefly, MCF-7 cells were treated with the compounds for 24 h followed by treatment with BP (0.5 μM). After 24h incubation, cellular DNA was isolated and analyzed for BP-derived DNA adducts by 32P-postlabeling technique. Manzamine A and sarcophine increased the BP-DNA adducts by 2 to 4-folds. Further, manzamine A (50 μM) substantially down regulated the expression of p53 while sarcophine (50 μM) slightly induced the level of p21. The residual DNA repair ability was almost completely abolished by manzamine A while sarcophine was ineffective. Based on our preliminary results, these compounds may be classified as potential genotoxic.

Post-irradiation DNA synthesis inhibition and G2 phase delay in radiosensitive body cells from non-Hodgkin's lymphoma patients: an indication of cell cycle defects

In the present study, both post-irradiation DNA synthesis and G2 phase accumulation were analyzed in lymphoblastoid cell lines (LCLs) and fibroblast cell strains derived from (Saudi) patients with non-Hodgkins lymphoma (NHL), ataxia telangiectasia (AT), AT heterozygotes and normal subjects. A comparison of the percent DNA synthesis inhibition (assayed by 3H-thymidine uptake 30 min after irradiation), and a 24 h post-irradiation G2 phase accumulation determined by flow cytometry placed the AT heterozygotes and the NHL patients in an intermediate position between the normal subjects (with maximum DNA synthesis inhibition and minimum G2 phase accumulation) and the AT homozygotes (with minimum DNA synthesis inhibition and maximum G2 accumulation). The similarity between AT heterozygotes and the NHL patients with respect to the two parameters studied after irradiation was statistically significant. The data indicating a moderate abnormality in the control of cell cycle progression after irradiation in the LCLs and fibroblasts from NHL patients may explain the enhanced cellular and chromosomal radiosensitivity in these patients reported by us earlier. In addition to demonstrating a link between cell cycle abnormality and radiosensitivity as a possible basis for cancer susceptibility, particularly in the NHL patients, the present studies emphasized the usefulness of the assay for 24 h post-irradiation G2 phase accumulation developed by Lavin et al. (1992) in characterizing AT heterozygote-like cell cycle anomaly in cancer patients irrespective of whether they carried the AT gene or any other affecting the cell cycle.

Reduced induction of p53 protein by γ-irradiation in ataxia telangiectasia cells without constitutional mutations in exons 5, 6, 7, and 8 of the p53 gene

Ataxia telangiectasia (AT) is an autosomal recessive disease of childhood with several phenotypic characteristics. One of the hallmarks of this syndrome is its hypersensitivity to ionizing radiation, which is believed to be due to defects in DNA repair/processing. In addition to radio-resistant DNA synthesis, both fibroblasts and lymphoblastoid cell lines derived from these patients have been shown to have an impaired G1 arrest and prolonged G2 accumulation of cells indicating a defect in the regulation of cell cycle after irradiation. Since the (tumor suppressor) p53 protein has been reported to participate in the regulation of G1 arrest after irradiation, the possibility of p53 gene mutation and deregulating cell cycle in AT needed to be examined. We used the PCR amplification and DNA sequencing methods to detect mutations in the hypermutable exons (5-8) of germline p53 in fibroblast cells from 3 AT homozygotes. No mutation was found in any of these exons. In order to determine the role of the p53 protein in G1 arrest, its levels were measured before and after gamma-irradiation by flow cytometry in both AT and normal cells. Radiation-induced p53 protein levels in the AT cells varied from 6 to 60% compared to the normal cells, indicating a reduced induction of the protein in AT. These results suggest that mutation in the AT gene affects the p53 induction by irradiation and may, thus, alter the cell cycle regulation in the AT patients.

Cellular radiosensitivity of patients with papillary thyroid cancer

Cultured skin fibroblasts from patients with papillary (differentiated) thyroid carcinomas were compared to those from healthy subjects, ataxia telangiectasia (AT) homozygotes and AT heterozygotes for colony-forming ability after low dose-rate irradiation, and post-irradiation DNA synthesis as indicated by uptake of [3H]-thymidine. The cells from the cancer patients exhibited enhanced radiosensitivity (intermediate between normal and AT) and less than normal level of radioinduced inhibition of DNA synthesis.

Chronic γ-irradiation results in increased cell killing and chromosomal aberration with specific breakpoints in fibroblast cell strains derived from non-Hodgkin's lymphoma patients

Abstract Cultured skin fibroblast cells from 6 patients with non-Hodgkins (NHL) and 2 clinically normal subjects were compared for cell survival and chromosomal aberration after chronic γ-irradiation. Fibroblasts from an ataxia telangiectasia (AT) homozygote and an AT heterozygote were used as positive controls. Following irradiation, fibroblasts from all 6 NHL patients showed an increase in both cell death and chromosomal aberration (breaks and rearrangements) compared to the normal subjects. The difference in the frequency of chromosomal aberration between the normals and the NHL patients remained virtually unchanged over a period of 24–72 h post irradiation incubation of the cells. Cell cycle analysis by flow cytometry carried out in 1 normal and 1 NHL fibroblast cell strain showed that more cell representing the NHL patient were in G 2 /M phase compared to the normal at various times of cytogenetic analysis. While the AT homozygote appeared to be the most radiosensitive, the AT heterozygote showed a slightly higher incidence of cell death and chromosomal aberration than the normals. The cellular and chromosomal radiosensitivity of fibroblast cell lines from the NHL patients differed slightly from that of the AT heterozygote but clearly occupied an intermediate position between the AT homozygote and the normal subjects. Cells from 3 of the NHL patients showed radiation-induced specific chromosomal breaks involving chromosomes 1, 2, 6, 8, 10 and 11 which correspond to known fragile sites. Such breakpoints associated with increased radiosensitivity may be indicative of predisposition to malignancy in the patients studied.