Sister chromatid exchange levels and cell cycle time in human bone marrow cells and lymphocytes

The frequency of sister chromatid exchange (SCE) and the cell-cycle-specific pattern of mitoses were analyzed at the same time in normal bone marrow cells and lymphocytes of six healthy donors. The SCE frequency was found to be significantly higher in lymphocytes. The cell-cycle-specific pattern revealed significantly shorter cell cycle times for normal bone marrow cells as compared with those of phytohemagglutinin (PHA) stimulated lymphocytes. Chromosomes of bone marrow metaphases displayed a more contracted morphology.     

Age-related variation in sister chromatid exchanges and cell cycle kinetics in peripheral blood lymphocytes of healthy individuals

Sister chromatid exchanges (SCEs) and cell cycle kinetics were estimated in mitogen stimulated human lymphocytes from a selected group of healthy individuals. Data were examined to evaluate the relationship between SCE frequencies and cell cycle kinetics with donor's age, sex and smoking habit. No regular relationship was observed between the means SCE frequencies and donor's age, although significant differences were observed between the age groups. Correlation of dispersion coefficient (H) of SCE with donor's age were significant in male and female populations. For cell cycle kinetics, a highly significant age-dependent depression in replicative index (RI) was observed. Female donors possessed a slightly higher SCE frequency and RI, although the variations between the two sexes were not significant. Smoking habit resulted in a significant enhancement of SCEs.     

Growth rate and sister chromatid exchange (SCE) incidence of bone marrow cells in Acute Myeloblastic Leukemia (AML)

The sister chromatid exchange (SCE) incidence and growth kinetics have been studied by means of an in vitro bromodeoxyuridine (BrdU) chromosome labeling method in the bone marrow cells of 17 acute myeloblastic leukemia (AML) patients with only diploid cells at diagnosis, remission, and relapse of the disease. At diagnosis, the cells tended to exhibit a low SCE frequency as compared to that during remission. An increased SCE frequency was observed after chemotherapy during remission or relapse. At diagnosis and relapse, when leukemic blast cells predominated in the marrow, they were characterized by the predominance of cells that had undergone only one cell cycle after BrdU exposure. In contrast, the marrow cells during remission tended to resemble the control pattern of growth kinetics, with a predominance of cells undergoing second and third cell cycles in the presence of BrdU. These results suggest that the growth rate of leukemic and nonleukemic cells is different, and that chemotherapy can cause an increased SCE frequency in the marrow cells of AML patients irrespective of the state of the disease.     

Chromosomal breakage and sister chromatid exchange in peripheral blood lymphocytes and lymphoblastoid cell lines in the X-linked lymphoproliferative syndrome

The frequencies of chromosomal aberrations and sister chromatid exchange (SCE) were measured in lymphoblastoid cell lines (LCLs) and in phytohemagglutinin (PHA)- and pokeweed mitogen (PWM)-stimulated lymphocytes from males with X-linked lymphoproliferative (XLP) syndrome, their obligate carrier mothers, and control subjects. We observed an increased frequency of chromosomal aberrations including increased polyploidy in LCLs derived from families with XLP with time in culture. The SCE rate in LCLs (mean of 3.89 SCEs per cell) was much lower than that in PHA- or PWM-stimulated lymphocytes: PWM-stimulated lymphocytes showed 9.58 SCEs per cell and PHA-stimulated cells had 11.38 SCEs per cell. A greater number of chromosomal gaps and breaks in the D-group chromosomes of LCLs of affected males and carrier females were identified compared to the number expected, based on chromosomal length and the number of aberrations seen in PHA-stimulated cell cultures. No differences in the frequency of SCEs or chromosomal aberrations were found in control subjects and affected males or carrier females in the peripheral lymphocytes stimulated by PHA. Phenotypes of XLP appear to arise from failure of immune responses to Epstein-Barr virus (EBV) and not from intrinsic chromosomal breakage or instability.     

Growth of pre-B cells in cultures of bone marrow from children with acute lymphoblastic leukaemia and other diseases.

Pre-B cells from the bone marrow of children with acute lymphoblastic leukaemia (ALL) survived up to 144 hr after the completion of treatment and divided in culture with maximum cell numbers at 24 hr. There was no rise in B cell number and no evidence of differentiation from pre-B to B cells. Binucleated pre-B cells in cultures containing cytochalasin B confirmed that pre-B cell division was occurring. Cycloheximide reduced cell numbers in culture but bromodeoxyuridine did not. Pre-B cell numbers also increased in culture of morphologically normal marrows from treated and untreated patients with solid tumours, and probably in normal marrows from patients with non-malignant diseases.     

Cytogenetic and cytokinetic analysis of lymphocytes from patients with hereditary adenomatosis of the colon and rectum

The frequencies of chromosome aberrations, sister chromatid exchanges (SCEs), and cell cycle kinetics were examined in cultured lymphocytes from five patients with hereditary adenomatosis of the colon and rectum (ACR) [three patients with Gardner's syndrome (GS) and two patients with familial polyposis coli (FPC)]. The frequency of numerical chromosome aberrations was no different in metaphase cells at the first and second replication cycles (M1 and M2) in the ACR patients and control subjects. The percentage of structural chromosome aberrations in both M1 and M2 cells was somewhat higher in the ACR patients as compared with the controls. Neither spontaneous nor mitomycin C-induced SCE frequencies in the patients with ACR were different from the controls, except for one patient with GS, who showed a remarkably high spontaneous SCE frequency. This patient is the mother of a son who had hepatoblastoma. The cell replication index (RI) was lower in the GS patients than in the controls. However, the RI in the FPC patients did not differ from that of the controls.     

Sister chromatid exchange with and without in vitro mutagen induction in cultured skin fibroblasts from patients with adenomatosis of the colon and rectum

Fibroblast cell strains were obtained from skin biopsies taken from patients with adenomatosis of the colon and rectum (ACR), and their relatives. A total of 50 different fibroblast strains were tested for their frequencies of sister chromatid exchange (SCE) in vitro. These strains included nine from patients with the Gardner syndrome, 21 from patients with non-Gardner ACR, and 20 cell strains from healthy relatives who were not at an increased risk for ACR. In 23 strains, the SCE frequencies after in vitro exposure to N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) were also determined. Both with and without MNNG induction, SCE values in the Gardner strains were found to be significantly higher than in the control strains (p less than 0.02 and p less than 0.03, respectively). Non-Gardner ACR strains differed only slightly from controls, thus making the difference between the control group and the pooled Gardner + non-Gardner ACR group not significant. In all groups, there was a significant increase in SCE after MNNG exposure, and those strains which had low SCE values spontaneously, also tended to have relatively moderate SCE values after MNNG induction. There was no significant difference between the ratios of SCE values with and without MNNG exposure in the different groups.     

A comparison of baseline and cyclophosphamide-induced sister chromatid exchanges in bone marrow and spleen cells of mouse and Chinese hamster

Baseline sister chromatid exchange (SCE) frequencies were investigated in bone marrow and spleen cells of mice and Chinese hamsters. No significant difference in SCE frequency was noted for bone marrow in both species and for bone marrow and spleen in mice on per cell and per pg DNA basis. However, a significant difference was noted between species in spleen and between cell types in Chinese hamsters. Also, statistically significant differences were noted between species for both cell types when the same data were expressed on per chromosome basis. SCE levels in cultured bone marrow and spleen cells after intraperitoneal administration of the antineoplastic drug cyclophosphamide (10 and 20 mg/kg) differed significantly in mice and Chinese hamsters on per cell, per pg DNA content, and per chromosome basis. The spleen cells were much more sensitive to the effects of cyclophosphamide than bone marrow cells in both species. The replicative indices did not differ significantly between treated and control animals in either bone marrow or spleen cells of both species. Since SCE frequency is a sensitive measure of DNA damage, and bone marrow and lymphocytes are the most widely used cell types in human and animal in vivo assays, the methodologies and results reported here may be useful for comparative mammalian cytogenetic studies.     

Sister chromatid exchanges in leukemic patients

Sister chromatid exchange (SCE) was studied in PHA-stimulated peripheral blood lymphocytes from 36 newly diagnosed and untreated leukemic patients: 16 with acute lymphoblastic leukemia (ALL), 10 with acute nonlymphocytic leukemia (ANLL), and 10 with chronic myelocytic leukemia (CML). The metaphases analyzed show no chromosomal abnormalities. The mean SCE frequency (mean +/- SE) for each group of patients was: 6.8 +/- 0.4, 6.6 +/- 0.3, and 7.0 +/- 0.6 per mitosis, respectively, which was significantly lower than the mean SCE score for 30 controls (8.7 +/- 0.2). No differences in SCE score among ALL, ANLL, and CML and a similar SCE frequency by chromosome number and group allowed consolidation of all the cases into a single group of 36 leukemic patients (6.8 +/- 0.3). When the frequency of SCE was compared by chromosome number and group between the leukemic patients with the control group, a significant decrease in SCE frequency was observed due to a low SCE score in almost all the complements, except chromosome #1. It is suggested that the low SCE rate is related to the leukemic process itself.     

Granular acute lymphoblastic leukaemia of childhood: a morphological phenomenon.

Three hundred and twenty consecutive children with lymphoblastic leukaemia (ALL), treated on the Medical Research Council UKALL VIII schedule, had their Romanowsky stained diagnostic marrows reviewed for the presence of azurophil granules in blast cell cytoplasm. Twenty patients (7%) had greater than 5% blasts showing this feature; 19 had the cell phenotype of "common ALL." Male children and those with French-American-British (FAB) L2 morphology predominantly showed this feature. There was also a strong correlation between granularity and non-diffuse acid phosphate positivity, but no obvious difference between the 20 patients in their response to treatment emerged during a minimum follow up of 15 months. The "granular" variant occurs in around 7% of children with ALL, but has no clear prognostic importance. Morphologists should be aware of its existence and incidence to avoid confusion with acute myeloid leukaemia.     

Sister chromatid exchanges, chromosome aberrations and micronuclei in female lymphocytes: correlations with biological rhythms, miscarriages and contraceptive pill use

Our study looked at the variation in peripheral blood lymphocytes, during the menstrual cycle, of frequencies of sister chromatid exchanges (SCE) and micronuclei (MN) in 819 women and cells with aberrant chromosomes (CA) in a selected sample of 136 volunteers. We observed significant fluctuations in SCE and CA frequencies: SCEs reached a maximum value at the end of menstruation and a low at the time of ovulation, whereas CAs showed a continuous increase from the beginning of the menstrual cycle up to the time of ovulation and a progressive decrease thereafter. MN frequency did not fluctuate in a statistically significant way. No statistically significant differences in SCE, CA and MN frequencies were observed when fertile women were compared with women taking the contraceptive pill or those in menopause and no difference was found between women who had undergone physiological or surgically induced menopause. Moreover, no difference was found between women with a history of miscarriages and matched controls. These data together suggest that the natural variations in sexual hormone levels, but not those due to the contraceptive pill or their reduction at menopause, can contribute in modulating the baseline frequencies of SCEs and CAs. Moreover, these data suggest that the increased risks either of producing a chromosome imbalance in the progeny (eliciting miscarriages) or of occurrence of gynaecological diseases is not predictable by evaluating cytogenetic end-points in peripheral blood lymphocytes.     

Malignant melanoma: sister chromatid exchange analysis in three families

Sister chromatid exchange (SCE) was analyzed in stimulated lymphocytes and skin fibroblasts in members of three families with cutaneous malignant melanoma (CMM). Two of these families were characterized by familial CMM; the other family had one patient affected by CMM and two others with other cutaneous melanocytic lesions. All the patients had undergone surgery but no chemotherapy. Higher and differing SCE rates were found in lymphocytes and in fibroblasts of all patients. A wide range of SCE distribution was found in patients with high SCE rate. A few healthy close relatives also showed relatively high SCE rates and wide range distributions. These subjects may be regarded as a subset of family members at high risk for developing cancer. The variability of SCE rates and distribution may reflect genetic heterogeneity of CMM.     

Induction and reduction of sister chromatid exchange by CCNU in human lymphocytes in vitro

Sister chromatid exchange (SCE) was studied in human lymphocytes treated with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in vitro. A dose-dependent increase of SCE was observed in cells exposed to 10(-5) - 10(-4) M CCNU. The maximal increase was 25-35 SCEs/cell over the control level, which is similar to the increase found in patients treated with CCNU in vivo. In the presence of rat liver microsomes (S-9 fraction) the frequency of CCNU-induced SCE was slightly higher than in parallel cultures without S-9, suggesting that microsomal metabolism may enhance the rate of decomposition of CCNU into reactive products. The CCNU-induced increase of SCE was greater in cells treated for longer time periods (up to 70 hr) than in cells subjected to a 1-hr treatment. This effect was most pronounced at higher concentrations of the drug (5 X 10(-5) M). The frequency of CCNU-induced SCE was also found to be dependent on the time of treatment in the cell cycle. A treatment for 1 hr during early G1-phase (about 20 hr before the first S-phase) gave rise to a higher increase of SCE than a 1 hr treatment immediately before or during the first or second S-phase. Thus, the CCNU-induced DNA damage leading to SCE seems to persist and may even increase during the prereplicative phase of the cell cycle. After replication in BrdUrd-free medium, the frequency of CCNU-induced SCEs decreased to the control level. The present results, taken together with other studies of strand break and cross-link formation by CCNU in mammalian cells in vitro, suggest that the major SCE-inducing damage by CCNU is DNA interstrand cross-links. These lesions then appear to be slowly removed, if at all, during the prereplicative phase of the cell cycle, and to disappear during or after replication in BrdUrd-free medium in vitro.     

In vivo persistence of sister chromatid exchanges (SCE) induced by gamma rays in mouse bone marrow cells

The sister chromatid exchange (SCE) frequencies induced in bone marrow cells by in vivo irradiation with gamma rays before or after bromodeoxyuridine (BrdUrd) incorporation were compared. The frequency of SCE at different postirradiation times was also measured in bone marrow cells in vivo, irradiated before BrdUrd incorporation. Increased sensitivity to SCE induction by radiation was found in cells after BrdUrd incorporation for one cycle when compared with cells irradiated before BrdUrd incorporation. The increased SCE frequency persisted for at least 72 hr after the initial irradiation, implying that the gamma ray-induced lesion(s) capable of eliciting an SCE are persistent and cannot be easily repaired.     

Distinctive cytoplasmic surface activity in benign and leukemic bone marrow cells: ultrastructural definition and clinicopathologic correlation

A distinctive ultrastructural finding, observed in a case of acute lymphoblastic leukemia (ALL) and previously unreported on the surface of bone marrow cells, is described. The alteration is referred to as distinctive cytoplasmic surface activity (DCSA). For further delineation, study of 130 consecutively submitted bone marrow specimens was undertaken. All samples were obtained from children and included diverse myeloid leukemias, acute lymphoblastic leukemia, normal marrows, and abnormal but non-neoplastic conditions. The DCSA was composed of simple and complex interconnected tubular elements in continuity with the cytoplasmic membrane, probably, sharing its glycocalyx. The intensity of the DCSA varied but, its presence was detected in one third of the 130 cases. Except for rare marrows (three cases) with DCSA bearing polymorphs, all positive cells were lymphoid in origin, usually originating from non-B, non-T ALL patients. In very few non-neoplastic cases, DCSA-positive lymphocytes also were found. Review of the literature shed little light on the subject but disclosed rare instances in which DCSA was illustrated in benign as well as malignant lymphoid cells. Based on this information and the observations made throughout the study, it is surmised that DCSA may be the morphologic expression of a cellular function, probably a form of micropinocytosis. Despite its prevalence in ALL cases, it appears to be nondiagnostic and nonspecific to a cell line.     

Effects of acute and chronic administration of mitomycin C on the induction of sister chromatid exchanges in vivo

Frequencies of sister chromatid exchanges (SCE) were analyzed in bone marrow cells of mice injected with mitomycin C (MMC) both before and during infusion with bromodeoxyuridine (BrdU). Administration of MMC at 1, 6.5, and 13 hours after the onset of BrdU infusion resulted in the induction of approximately 45 SCE/cell, independent of time of administration. When MMC was injected 26 hours prior to BrdU infusion, only baseline levels of SCE were noted. The effects of multiple doses of MMC (chronic administration) were examined in mice treated with 1–5 mg/kg on a weekly or bimonthly basis. SCE analysis was performed one week after the final injection. At all doses and with all treatment regimes, SCE frequencies did not differ from control levels. The results indicate that most or all MMC-induced DNA damage that results in SCE formation is removed in a single cell cycle after its administration.     

Phorbol-12,13-dibutyrate improves the quality of cytogenetic preparation in lymphoid malignancies

In cytogenetic preparation of lymphoid malignancies we investigated the quantitative and qualitative impact of phorbol-12,-13-dibutyrate (P) and of this tumor promoter in combination with the calcium ionophore A23187 (PA). Using parallel cultures of unstimulated and stimulated preparations, the effect was examined in 13 patients with malignant lymphomas and six patients with acute lymphoblastic leukemias (ALL). Focusing on high-quality analyzable metaphases, the best results were found in seven of 13 cases with lymphomas and five of six patients with ALL in the cultures supplemented with phorbol-12,13-dibutyrate. The yield of metaphases of good quality regarding length, spreading, and banding of chromosomes was regularly better in F-stimulated 24-hour culture (p < 0.05), followed by 48-hour cultures stimulated with P alone. Addition of the calcium-ionophore was of no further benefit. The yield of the unstimulated direct harvest was rather poor in nearly all patients investigated. Because no mutagenic effect of P was observed, the use of this mitogen may offer interesting perspectives in cytogenetic analysis of lymphoid malignancies and perhaps also in other tumors with low mitotic indexes.