Probing the pathobiology of response to all-trans retinoic acid in acute promyelocytic leukemia: premature chromosome condensation/fluorescence in situ hybridization analysis.

The response of acute promyelocytic leukemia (APL) peripheral blood and bone marrow cells to trans-retinoic acid (RA) was cytogenetically characterized during RA treatment using the techniques of premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH). Before treatment, the predominant immature bone marrow cells were found to have t(15;17), whereas the residual mature granulocytes were diploid and lacked evidence of the translocation. In response to RA treatment, an increase in the leukocyte count was noted. The majority of these cells exhibited a t(15;17). Subsequently (eg, between days 6 and 23), 32% to 91% of the maturing myeloid cells still exhibited t(15;17). The appearance of t(15;17) in gradually maturing elements suggests that RA contributed to a release of the maturation block of the leukemic elements. As responding patients obtained complete remission, diploid elements without evidence of the translocation prevailed in the blood and bone marrow. In 16 patients studied after 1 month in complete remission, all but 2 showed all diploid cells. The residual t(15;17) cells disappeared 18 days later in 1 patient, whereas the second patient exhibited clinical evidence of relapse 20 days later. These results suggest that response of patients with APL to RA is associated with maturation, subsequent loss of the mature leukemic elements, and preferential regeneration of normal diploid hematopoietic elements.

G2 radiosensitivity of cells derived from cancer-prone individuals.

The potential of enhanced chromatid damage, observed after X-irradiation of G2 phase, has been used to detect individuals genetically predisposed to cancer, utilising fibroblast/lymphocytes from these patients as well as fibroblasts derived from human tumours. Fibroblasts and/or lymphocyte samples of two autosomal recessive syndromes (xeroderma pigmentosum (XP), Fanconi's anaemia (FA)) and one congenital or acquired disorder, aplastic anaemia (AA), were employed for the G2 radiosensitivity assay. In addition, we have estimated the frequencies of spontaneously occurring chromosomal aberrations as well as G2 radiosensitivity of eight samples of fibroblasts/fibroblast-like cells (two normal, two colorectal carcinoma, two Wilms' tumour, one retinoblastoma and one polyposis coli), and three samples of lymphocytes (two normal and one from a lymphoma patient). The results obtained indicate that there were no differences between fibroblast cells derived from patients or tumours, except FA patients, in the frequency of spontaneously occurring chromosomal aberrations when compared to normal cells. Following X-irradiation we did not observe any significantly increased G2 radiosensitivity in FA and XP cells. Lymphocytes from AA and lymphoma patients, and all tumour cell lines except retinoblastoma, responded with increased frequencies of aberrations following G2 X-irradiation in comparison to cells derived from normal individuals. In our hands, the G2 sensitivity assay could not always discriminate cells from cancer-prone individuals from those of controls.

Chromosome breakage at a major fragile site associated with P-glycoprotein gene amplification in multidrug-resistant CHO cells.

Recent studies of several drug-resistant Chinese hamster cell lines suggested that a breakage-fusion-bridge mechanism is frequently involved in the amplification of drug resistance genes. These observations underscore the importance of chromosome breakage in the initiation of DNA amplification in mammalian cells. However, the mechanism of this breakage is unknown. Here, we propose that the site of chromosome breakage consistent with the initial event of P-glycoprotein (P-gp) gene amplification via the breakage-fusion-bridge cycle in three independently established multidrug-resistant CHO cells was located at 1q31. This site is a major chromosome fragile site that can be induced by methotrexate and aphidicolin treatments. Pretreatments of CHO cells with methotrexate or aphidicolin enhanced the frequencies of resistance to vinca alkaloid and amplification of the P-gp gene. These observations suggest that chromosome fragile sites play a pivotal role in DNA amplification in mammalian cells. Our data are also consistent with the hypothesis that gene amplification can be initiated by stress-induced chromosome breakage that is independent of modes of action of cytotoxic agents. Drug-resistant variants may arise by their growth advantage due to overproduction of cellular target molecules via gene amplification.

Frequencies of X-ray-induced chromosome translocations in human peripheral lymphocytes as detected by in situ hybridization using chromosome-specific DNA libraries.

In situ hybridization with chromosome-specific DNA libraries was used to analyse radiation-induced stable translocations in human peripheral blood lymphocytes. These data were compared with radiation-induced unstable-type aberrations (dicentrics) in the same samples. The results indicate that far more stable aberrations are induced by radiation in comparison to unstable aberrations.

PCR amplification of chromosome-specific DNA isolated from flow cytometry-sorted chromosomes.

We have established a method for amplifying and obtaining large quantities of chromosome-specific DNA by linker/adaptor ligation and polymerase chain reaction (PCR). Small quantities of DNA isolated from flow cytometry-sorted chromosomes 17 and 21 were digested with MboI, ligated to a linker/adaptor, and then subjected to 35 cycles of PCR. Using this procedure, 20 micrograms of chromosome-specific DNA can be obtained. Southern blot analysis using several DNA probes previously localized to chromosomes 17 and 21 indicated that these gene sequences were present in the amplified chromosome-specific DNA. A small quantity of the chromosome-specific DNA obtained from the first round of PCR amplification was used to amplify DNA for a second, third, and fourth round of PCR (30 cycles), and specific DNA sequences were still detectable. Fluorescence in situ hybridization using these chromosome-specific DNA probes clearly indicated the hybridization signals to the designated chromosomes. We showed that PCR-amplified chromosome 17-specific DNA can be used to detect nonrandom chromosomal translocation of t(15;17) in acute promyelocytic leukemia by fluorescence in situ hybridization.

Radiation-induced chromosomal breakage and rejoining in interphase-metaphase chromosomes of human lymphocytes.

The premature chromosome condensation (PCC) technique and conventional chromosome analysis were applied to examine the kinetics of radiation-induced primary chromosome breaks, their rejoining and formation of dicentrics in human peripheral blood lymphocytes (PBLs). Numbers of chromosomal elements and dicentrics per cell were analyzed for each dose. Dose-dependent increases were observed for chromosome fragments (linear) and dicentrics (linear/quadratic). For an assessment of repair kinetics, numbers of breaks and dicentrics were estimated immediately and at several recovery periods after irradiation, using the PCC technique. It was found that chromosome fragments restitute with time, whereas the dicentrics are formed very quickly and their frequency remains the same, despite the decline in the number of chromosome breaks at later recovery times. Fractionation experiments were conducted to study the time-dependent interaction of primary breaks in the formation of dicentric chromosomes. PBLs were irradiated with 3 Gy X-rays split into 2 equal fractions separated by different intervals up to 5 h. No marked difference was observed in the yield of dicentrics following the different fractionation protocols, except that the mean levels of dicentrics declined when the fraction interval was 4 h or more. It appears that most of the dicentrics are formed by misrepair of strand breaks, produced directly by radiation and not resulting from rejoining of existing breaks during the slow repair process which follows. We also studied the role of the chromatin configuration at the time of irradiation on the yield of chromosome fragments and dicentrics. Highly condensed chromatin due to pretreatment with 0.3 M NaCl was found to reduce the frequency of radiation-induced chromosomal aberrations.

A cytogenetic follow-up study of the victims of a radiation accident in Goiania (Brazil).

A radiation accident involving a cesium-137 therapy source occurred in Goiania (Brazil) in September 1987, in which more than 50 individuals were exposed to moderate to high doses (0.2-7 Gy) of gamma-radiation. A cytogenetic technique (i.e., frequencies of dicentrics and rings in peripheral lymphocytes) was employed to estimate the absorbed radiation dose. The follow-up study extending over more than 1 year indicated a decline in the frequencies of dicentrics in the lymphocytes. Using chromosome-specific biotinylated library probes for chromosomes 1, 2, 8 and 19, we studied the frequencies of chromosomal translocations and deletions and the incidence of aneuploidy in the lymphocytes of exposed individuals. In some individuals there was a significant increase in the frequency of translocations and aneuploidy. In other experiments, in which the frequencies of HPRT mutations were determined in lymphocytes using the BrdU-labeling method, some individuals showed an increase (from about 2- to 50-fold) in mutant frequencies.

Genotoxic effects of MeCCNU on human peripheral blood lymphocytes.

MeCCNU (semustine, 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea, NSC 95441) is successfully used in the treatment of various human malignancies. The drug was tested for its in vitro genotoxic effects on human peripheral blood lymphocytes. Several drug concentrations were studied considering mean plasma level achieved in patients after the receipt of MeCCNU therapy. Induction of chromosome aberrations and sister chromatid exchange frequency had shown dose-effect relationship. Reduced mitotic activity and prolonged average generation time was observed in MeCCNU treated cultures. The results suggest that 'therapeutic' MeCCNU concentrations have genotoxic effects on human peripheral blood lymphocytes.

Effects of CCNU therapy on human chromosomes.

Peripheral blood lymphocytes of 9 patients under CCNU therapy were examined for frequency of sister-chromatid exchanges (SCEs) and chromosomal aberrations (CAs). 7 out of 9 patients were treated with only CCNU, whereas the remaining 2 were treated with other chemotherapeutic agents in combination with CCNU. Compared to normal individuals, a significantly increased frequency of SCE was observed in the patients before starting anticancer therapy (P less than 0.001). Increased incidences of structural changes in chromosomes were observed in cells from all the treated patients. The most frequent aberrations were of chromatid type. After administration of a single dose of CCNU, an increase in SCE frequencies was observed which remained elevated even after 6 weeks. It was concluded that increases in SCEs and CAs in lymphocytes were caused by CCNU treatment. Further studies are needed to elucidate whether any CAs observed in the present study could participate in the induction of second neoplasm.

Complex translocation involving chromosomes #1, #9, and #22 in a patient with chronic myelogenous leukemia.

A case of Philadelphia chromosome positive chronic myelogenous leukemia with a complex translocation involving chromosomes #1, #9, and #22 is described. All cells in the bone marrow showed this rearrangement, and Q-banding analysis showed the predominant karyotype to be 46,XY, t(1;9;22)(p22;q34;q11).

Heteromorphism of C-band positive chromosomal regions in CML patients.

The heteromorphism of constitutive heterochromatin in chromosomes #1, #9, and #16 was investigated in 44 chronic myelocytic leukemia patients and 44 controls using bone marrow and peripheral blood lymphocyte cultures. A significant increase in the length of C-band region in all the three chromosome pairs as well as a statistically significant difference in the homologs of chromosome #1 was observed in chronic myelocytic leukemia patients when compared with the controls. The frequency of inversions was also greater in the patients than in the controls. A random translocation of 22q was found on either homolog of chromosome #9.

Genotoxic effects of ketamine on CHO cells.

Ketamine, a non-barbiturate anaesthetic agent, was studied for its genotoxic potential using the SCE assay. It was genotoxic in the in vitro system at concentrations comparable to the plasma levels achieved during steady state anaesthesia. It had no effects on cellular kinetics in CHO cells.

Cytogenetic effects of Me-CCNU on sister chromatid exchange frequency, cellular kinetics and chromosome aberrations.

1-(2-Chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (Me-CCNU) was tested for its in vitro effects on sister chromatid exchanges (SCE), cellular kinetics and chromosome aberrations in CHO cells. There was a relationship between the inhibitory activity of the drug and the cytogenetic damage, which was dose dependent. Increase in SCE values were highly significant (p less than 0.001) for all the four concentrations used. It also delayed the cell cycle progression. Inhibition of DNA synthesis results in increased frequency of chromosomal aberrations, which was highly significant for the higher concentrations, i.e. 5 micrograms and 10 micrograms Me-CCNU/ml.

Spontaneous and induced sister chromatid exchange frequencies and cell cycle progression in lymphocytes of patients with carcinoma of the uterine cervix.

Thirteen healthy females and thirteen untreated patients with carcinoma of the uterine cervix were studied for spontaneous and mitomycin C (MMC)-induced rates of sister chromatid exchange (SCE) and cell cycle progression. The mean values of spontaneous as well as MMC-induced SCE rates showed no statistically significant difference between groups. For studying cell cycle progression, cells in the M1, M2, and M3 stages were scored from the same samples. The percent values of cells in these stages, identified by the nature of differential sister chromatid staining, were found to be almost identical in normal as well as MMC-treated cultures in controls and patients. It was concluded that the presence of carcinoma of the uterine cervix in human females has no bearing either on spontaneous and MMC-induced SCE rates or on cell cycle progression in PHA-stimulated cultures of peripheral blood lymphocytes.