Mohammed A. Hannan

Mechanisms of cisplatin (cis-diamminodichloroplatinum II)-induced cytotoxicity and genotoxicity in yeast.

The antitumor drug, cisplatin (cis- diamminodichloroplatinum II), dissolved in both water and phosphate-buffered saline, was studied for its genotoxic and cytotoxic effects in the yeast, Saccharomyces cerevisiae. The results showed that the drug was both recombinagenic and mutagenic in the wild-type diploid strain D7. It was observed that both cytotoxicity and genotoxicity were greatly reduced when cisplatin was dissolved in phosphate-buffered saline compared to the aqueous solution. Cell survival analyses showed that the diploid strain (D7 rad 3), deficient in excision of UV-induced pyrimidine dimers or similar adducts, was hypersensitive to cisplatin. Another diploid strain (rad 52/rad 52), blocked in the repair of DNA double-strand breaks and recombination was also hypersensitive to the drug. Mitotic gene conversion was not observed in the rad 52/rad 52 diploid after the drug treatments, while it was reduced in the excision -deficient strain. Reverse mutations occurred in the excision-deficient strain (D7 rad 3), even at low doses of cisplatin. These results are discussed in relation to the possible mechanisms of cisplatin-induced cell death and genotoxicity.

Cytotoxicity of Khat (Catha edulis) extract on cultured mammalian cells: effects on macromolecule biosynthesis

Abstract A chloroform extract of Khat (Catha edulis) leaves was used to study the cytotoxic activity on KB, 1BR.3, and XP2Bi cells. Log phase cell survival curves showed an LD50 of 40 ng/ml for KB cells. 1BR.3 and XP2Bi cells were biphasic in their response to the extract during log phase, with an LD50 of 20 and 75 ng/ml, respectively. Stationary phase cells were unaffected by the extract. DNA and RNA synthesis inhibition was studied using radiolabeled thymidine or uridine to measure the amount of extract that inhibits the synthesis to 50% of the untreated control cells. DNA synthesis was inhibited by 45, 60 and 200 ng/ml and RNA synthesis by 24, 17 and 58 ng/ml in 1BR.3, XP2Bi and KB cells, respectively. Protein synthesis was inhibited to 15–20% of untreated control cells by a dose of 40 ng/ml in all the cells studied. From this work, it is apparent that the main cause of cytotoxicity of Khat extract may be the inhibition of de novo RNA synthesis. Our results suggest that this effect is exerted on all cells used in this study and that KB cells demonstrate a higher resistance to the toxic component.

Cytogenetic characterization of ataxia telangiectasia (AT) heterozygotes using lymphoblastoid cell lines and chronic γ-irradiation

SummaryLymphoblastoid cell lines (LCLs) derived from two patients identified as ataxia telangiectasia (AT), two obligate AT heterozygotes and two controls (healthy subjects with no known genetic disease or relationship to AT patients) were compared with respect to the induction of chromosomal breaks by acute and chronic γ-irradiation. Although there was a considerable increase in the frequency of chromosomal breaks per cell in the LCLs of AT patients resulting from acute irradiation, the small increase occurring in the LCLs of the AT heterozygotes made it difficult to distinguish them from the controls. Following chronic γ-irradiation, however, the frequency of chromosomal breaks per cell in the LCLs of the AT heterozygotes occupied a significantly distinct position from that of the controls. These observations suggested that the use of chronic irradiation may be a better choice in the cytogenetic characterization of AT heterozygotes.

Co-mutagenic effects of 2-aminoanthracene and cigarette smoke condensate on smoker's urine in the Ames Salmonella assay system.

Cigarette smoke condensate (CSC) and cigarette smokers urine (concentrated) (UC) were tested alone and in combination with direct and indirect mutagens for histidine reversion in the Ames Salmonella assay system. While both CSC and smokers urine showed some mutagenic activity upon metabolic activation with S9-mix, each of them in combination with the aromatic amine 2-aminoanthracene (2AA) exhibited a synergistic effect on mutagenicity. Such a synergistic effect was not found when these agents were combined with the direct mutagens, ethylmethane sulfonate (EMS) and methylmethane sulfonate (MMS), or the indirect mutagens, benzo[a]pyrene (BP) and 9,10-dimethyl-1,2-benzanthracene (DMBA), tested in this study, nor was the synergistic effect observed when 2AA was tested with urine from a non-smoker. The results, thus, reflected a specificity of the co-mutagenic action of factor(s) in cigarette smoke or smokers urine and 2AA. The significance of co-mutagens in carcinogenesis has been discussed and the importance of investigating co-mutagenesis particularly in the case of suspected human exposure to multiple environmental agents has been emphasized.

Mutagenicity of cisplatin and carboplatin used alone and in combination with four other anticancer drugs

Mutagenicity of cisplatin and carboplatin was compared by using the drugs alone and in combination with bleomycin, 5-fluorouracil, vincristine and methotrexate in the Ames Salmonella assay employing the tester strains TA98, TA100 (excision deficient) and TA102 (excision proficient). Cisplatin showed the maximum yield of histidine revertants in TA98 and TA100 at 2 micrograms/plate followed by a decrease in the number of mutants/plate with increasing concentrations. In the excision proficient strain TA102, there was no decline in the number of mutants/plate even at a concentration of 8 micrograms/plate. Basically, similar results were also obtained with carboplatin but using higher concentrations of the drug. When cisplatin or carboplatin was combined with other anticancer drugs, there was no differential modification of mutagenicity of the 2 platinum compounds in any of the bacterial tester strains.

A molecular study of EBV DNA and p53 mutations in nasopharyngeal carcinoma of Saudi Arab patients

Tumor biopsies obtained from 25 Saudi Arab patients with nasopharyngeal carcinoma (NPC) were examined for the presence of Epstein-Barr virus (EBV) DNA detected by the polymerase chain reaction (PCR) and for the incidence of p53 mutations screened by a combination of PCR, single strand conformation polymorphism (SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP). DNA sequencing was carried out to confirm the occurrence of p53 mutation. While 92% of the tumor specimens were found to carry EBV DNA, only 1/25 showed the incidence of a homozygous mutation at codon 248 of the p53 gene. The data showed that despite a high association of EBV infection with Saudi NPC, the frequency of p53 mutations was very low. Our results are consistent with the worldwide observation of infrequent p53 mutations in NPC.

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.

Ataxia-oculomotor apraxia syndrome.

Ataxia-oculomotor apraxia is a distinct entity first comprehensively described in 1988. The features include early childhood onset of ataxia and oculomotor apraxia, mimicking ataxia telangiectasia but without the extraneurologic findings of ataxia telangiectasia. We add to the clinical description of the ataxia-oculomotor apraxia syndrome by reporting eight patients, ages 2 to 15 years, from four families, suggesting autosomal recessive inheritance, with the longest follow-up over 6 years. After initial gait deterioration, all had a nonprogressive course. We have postulated that ataxia-oculomotor apraxia should be established as a separate disease from ataxia telangiectasia or its variants not only by clinical history, examination findings, and course, but primarily by the biologic markers of normal chromosome breakage and radiation sensitivity studies. We found no increased chromosome breakage in the four patients studied and intermediate sensitivity to chronic ionizing radiation of cultured skin fibroblasts on the three patients studied. Family studies revealed an intermediate radiosensitivity from two patients, their asymptomatic parents, and a sister. The lack of chromosome breakage strongly separates ataxia-oculomotor apraxia from ataxia telangiectasia. The radiation sensitivity studies are compatible with two possibilities: (1) symptomatic ataxia telangiectasia heterozygotes, but this would be highly unusual because the degree of clinical involvement in the ataxia-oculomotor apraxia patients is not mild, as would be expected if they were heterozygotes and (2) a separable disease entity, which is the interpretation we favor. (J Child Neurol 1995;10:118-122).

Cellular radiosensitivity of patients with different types of neurofibromatosis

Skin fibroblast cell strains were developed from nine Saudi patients with different types of neurofibromatosis (NF) and nine healthy subjects (controls), and their radiosensitivity was compared following chronic exposure to gamma-radiation at a dose rate of 0.0076 Gy/min (1 Gy = 100 rads). Cells from both normal appearing skin and café-au-lait spots of the different NF patients (7 out of 9) clearly showed increased radiosensitivity, with D10 (dose resulting in 10% survival) values of 2.0-4.4 Gy for the former and 3.0-4.8 Gy for the latter, compared to the normal controls (with D10 values of 6.1-10.6 Gy). These data provide further evidence of an association of enhanced cellular sensitivity to chronic irradiation with NF regardless of the classes they belong to. Hypersensitivity to specific carcinogens may, thus, be a factor responsible for the increased propensity to cancer in these patients.

Chronic γ-radiation sensitivity of skin fibroblasts from patients with non-Hodgkin's lymphoma (NHL)

Abstract Cultured skin fibroblasts from 11 patients with non-Hodgkins lymphoma (NHL), 3 with ataxia telangiectasia (AT), 3 AT heterozygotes and 6 healthy subjects were studied for impaired colony-forming ability upon chronic exposure to γ-radiation. A comparison of survival curves of the different cell lines revealed an AT heterozygote-like response (intermediate radiosensitivity) in 8 (73%) out of 11 NHL patients. These results suggested that the majority of the NHL patients may have an underlying abnormality of DNA repair.