Spontaneous sister chromatid exchange in normal bone marrow and Ph-positive chronic myelocytic leukemia

The frequency of spontaneous sister chromatid exchange was studied in normal marrow derived from 38 healthy donors and 40 untreated patients with chronic phase CML. The sister chromatid exchange frequency was significantly lower in the leukemic cells (range, 2.32 +/- 1.31 to 4.76 +/- 2.37 per metaphase; mean, 3.18 +/- 0.49) than in normal marrow (range, 2.36 +/- 1.44 to 5.54 +/- 2.24 per metaphase; mean, 3.92 +/- 0.72). The contraction status of chromosomes was comparable in normal and Ph-positive metaphases. The reduction of sister chromatid exchange in leukemic cells was seen in all chromosome groups. The analysis of cell cycle specific proliferation according to the typical staining patterns of metaphases due to the number of cell cycles during which bromodesoxyuridine was substituted, revealed longer cell cycle times for the leukemic cells.     

The effect of imatinib mesylate on patients with Philadelphia chromosome-positive chronic myeloid leukemia with secondary chromosomal aberrations.

PURPOSE AND EXPERIMENTAL DESIGN: The acquisition of secondary chromosomal aberrations in chronic myeloid leukemia (CML) patients with Philadelphia chromosome-positive (Ph+) karyotype signifies clonal evolution associated with disease progression to accelerated/blastic phase. Therefore, these aberrations are of clinical and biological importance. We identified 58 cases with secondary abnormalities in addition to t(9;22)(q34;q11.2) or its variants in a review of 137 CML patients treated with imatinib mesylate (STI571). Clinically 13 patients were in chronic phase, 24 in accelerated phase, and 21 in blastic phase. RESULTS: More than 50% of cases showed the major routes of CML clonal evolution [+8, i(17q), +Ph, and/or +19]. The remainder exhibited minor routes of secondary abnormalities with -17/17p-, 11p-/rearr(11p), and -7/rearr(7) as the most frequent abnormalities. Of particular interest, one case developed an inv(16)(p13q22) as a secondary anomaly during blastic transformation. The bone marrow was consistent with myelomonocytic morphology with eosinophilia. Cytogenetic responses to imatinib mesylate occurred in 15 of 58 (26%) patients; 12 achieved complete cytogenetic remission, 2 had a major response, and 1 had a minor response, with most responses noted within 3-6 months. Seven patients remain in remission >17-30 months, 2 patients relapsed between 12 and 19 months on therapy, and 1 patient was treated by bone marrow transplantation. CONCLUSIONS: Although some Ph+ CML patients with clonal evolution can have a complete cytogenetic response to imatinib mesylate, responses tend to be brief, and such patients may benefit from subsequent stem cell transplantation. Therefore, CML patients with clonal evolution may require therapy additional to imatinib mesylate for maximal eradication of the Ph+ leukemic cells.     

Induction of sister chromatid exchanges and chromosome aberrations in cultured mammalian cells by N-Nitrosocimetidine

N-Nitrosocimetidine (NC) induces significant numbers of sister chromatid exchanges (SCE) and chromosome aberrations in cultured Chinese hamster ovary (CHO) cells even at a concentration of 1.2 × 10^?7 M. Its effectiveness in SCE induction is about two thirds that of the gastric carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). These results constitute further evidence that NC possesses carcinogenic activity.     

附加染色体异常的费城染色体阳性慢性粒细胞白血病患者临床特征及预后分析

目的:探讨附加染色体异常的费城染色体（Ph）阳性慢性粒细胞白血病（CML）患者的临床特征及预后。方法:回顾性分析2009年1月至2019年1月于青岛大学附属医院诊断的351例Ph + CML患者资料,患者均经R显带技术进行骨髓染色体核型分析。总结初诊时附加染色体异常Ph + CML患者临床特征及核型,采用Kaplan-Meier法分析不同核型患者总生存（OS）差异。 结果:351例Ph + CML患者中,32例（9.1%）为变异易位。初诊时附加染色体异常47例,包括慢性期29例、加速期3例和急变期15例,分别占全组慢性期的9.15%（29/317）、加速期的25.00%（3/12）和急变期的68.18%（15/22）,各期附加染色体异常发生率差异有统计学意义（ χ2=50.799, P<0.05）;47例附加染色体异常的Ph + CML患者中,13例为附加3种以上染色体异常的复杂核型,慢性期、加速期、急变期复杂核型比例分别为13.79%（4/29）、33.33%（1/3）、53.33%（8/15）,差异有统计学意义（ χ2=9.26, P<0.05）。慢性期患者中最常见的附加染色体异常为双Ph（48.28%,14/29）、-Y（10.34%,3/29）,急变期患者中最常见的为+8（26.67%,4/15）、双Ph（26.67%,4/15）。Kaplan-Meier生存分析显示,Ph + CML患者中,初诊时附加染色体异常者OS较非异常者差（ χ2=61.138, P<0.05）;初诊时附加染色体异常的Ph + CML患者中,复杂核型者OS较非复杂核型者差（ χ2=4.945, P<0.05）。 结论:附加染色体异常与CML疾病进展密切相关;附加染色体异常的Ph + CML患者的预后较只有Ph易位的患者差。附加染色体越复杂,CML急变的可能性越大,预后也越差;在CML治疗过程中出现附加染色体异常也可能导致急变的进展。     

Interphase FISH for follow-up of Philadelphia chromosome-positive chronic myeloid leukemia treatment

BACKGROUND: Several attempts have been made to determine whether interphase fluorescence in situ hybridization (I-FISH) on bone marrow or peripheral blood specimens is a good alternative to conventional cytogenetics (CC) in calculating the residual proportion of Philadelphia (Ph) chromosome-positive cells during treatment follow-up of patients with chronic myeloid leukemia. MATERIALS AND METHODS: Nineteen patients were selected for I-FISH follow-up compared to CC. All samples were also classified into 4 groups according to the percentage of residual Ph chromosome-positive metaphases analyzed in CC. I-FISH was performed using the LSI bcr/abl dual ES color probe (Vysis). RESULTS: A high correlation was observed between the frequency of Ph chromosome-positive cells, assessed by CC and I-FISH (p<0.001). A high correlation was found between CC and I-FISH for 12 patients, but not for the remaining 7. Applying the same classification for I-FISH did not show a good relationship between the two techniques (p<0.001). CONCLUSION: Dual color I-FISH is a reliable method to monitor the size of the Ph chromosome-positive clone in bone marrow of treated CML patients. However, it has to be complementary to conventional cytogenetics because it cannot detect the emergence of other chromosomal abnormalities in Ph chromosome-positive or -negative cells.     

Basophils exhibit rearrangement of the bcr gene in Philadelphia chromosome-positive chronic myeloid leukemia

Basophils were isolated from the peripheral blood of two patients with Philadelphia positive-chronic myeloid leukemia using monoclonal antibody Bsp-1 and fluorescence activated cell sorting. DNA blot analyses demonstrated rearrangement of the breakpoint cluster region gene in the isolated basophils, which suggests their leukemic origin. Isolated T cells from these patients that were cultured for 14 days in the presence of interleukin-2 lacked rearrangement of the breakpoint cluster region gene and are therefore unlikely to be derived from the chronic myeloid leukemia clone.     

Sister chromatid differentiation and cell-cycle-specific patterns in chronic myelocytic leukemia and normal bone marrow

After In vitro incubation with bromodeoxyuridine (BrdUrd)-containing medium, metaphases of Philadelphia (Ph<1>)-chromosome positive cells in the blood and/or bone marrow of eight untreated patients with chronic myelocytic leukemia (CML) were analysed by means of sister chromatid differentiation (SCD) and compared to 10 normal bone marrows. The majority of proliferating Ph<1>-positive cells (range: 46-86%) were unable to complete more than one cell cycle during the culture period of 50 h (M1 metaphases). Ph<1>-positive M2 metaphases which had completed two cell cycles were found in the range of 18-54% and almost no M3 metaphases were detected after passage through three cell cycles (0-1%). The corresponding findings in proliferating normal bone-marrow cells were: M1 (13-68%), M2 (30-79%) and M3 (0-18%). These data support the notion that there is a prolonged cell cycle time in CML which can be measured by SCD, demonstrating a proportional shift from M3/M2 to M1 metaphases. SCD is suggested for kinetic studies on human malignant cell clones characterized by marker chromosomes.     

Induction of sister chromatid exchanges by extremely low doses of alpha-particles.

The induction of sister chromatid exchanges (SCE) was examined in Chinese hamster ovary cells irradiated in the G1 phase of the cell cycle with alpha-particles from a plutonium-238 source. A significant increase in the frequency of SCE occurred with doses as low as 0.31 mGy (31 millirads). Although 30% of the cells showed an increased frequency of SCE at this dose, less than 1% of cell nuclei were actually traversed by an alpha-particle. A dose of approximately 2.0 Gy was necessary to produce a similar increase in SCE by X-rays. These results indicate that genetic damage may be induced by low doses of alpha-radiation in cell nuclei not actually traversed by an alpha-particle. This phenomenon may have important implications in the estimation of risks of such exposures.     

Sister chromatid exchanges and chromosomal aberrations in mouse cells infected with the Abelson and Moloney leukemia viruses

‘Spontaneous’ and mitomycin C (MMC)-induced sisterchromatid exchanges (SCE) and chromatid breaks were scored inANN-1 fibroblasts, a non-producer mouse cell line transformedby the Abelson murine leukemia virus (AMuLV), a replicationdefective retrovirus whose genome contains the v-abl oncogene.Normal, non-transformed NIH3T3 fibroblasts were used as control.SCE and chromatid break frequencies in untreated or MMC-treatedANN-1 and NIH3T3 cells were compared with those observed inthe same cells after infection with the helper murine Moloneyleukemia virus (M-MuLV), which rescues the ability of A-MuLVto replicate in ANN-1 cells. The frequency of spontaneous andMMC-induced SCE were not significantly different in both ANN-1and NIH3T3 cells, independently of M-MuLV infection. After M-MuLVinfection, however, increased ‘spontaneous’ frequencyof SCE and altered susceptibilty to the induction of SCE byMMC was observed in both cell lines compared to M-MuLV-uninfectedcells. In the case of chromatid breaks, the baseline frequencywas not significantly different between the two cell lines bothin the presence or in the absence of M-MuLV infection, nor wasit significantly increased by M-MuLV, with respect to the valueobserved in uninfected cells. These results indicate that, atvariance with what occurs with SCE, viral replication is notneeded to increase the frequency of chromosomal aberrationsand that the portion of A-MuLV genome alone is sufficient to increase chromatid breaks but not SCE in ANN-1 cells. Thus,in mouse cells carrying retroviruses, SCE and chromosomal aberrationsseem to be independently generated, and influenced by differentviral genes.     

Cytogenetic events after bone marrow transplantation for Philadelphia chromosome positive chronic myeloid leukemia

Allogeneic bone marrow transplantation is the only way to cure patients with Ph1+ chronic myeloid leukemia. It is commonly assumed that, in order to obtain a cure for the patients, the leukemic clone must be completely destroyed by the conditioning treatment and the donor's bone marrow must repopulate the hemopoietic niches leading to a "complete chimera". However, cytogenetic analyses, supported by molecular ones, indicate that Ph1+ cells, far from being completely destroyed by chemo-radiotherapy may persist for a long time, probably in the majority of the patients. As demonstrated by the outcome of patients receiving T-cell depleted marrow, immune mechanisms must be involved in controlling and progressively reducing the size of the residual leukemic clone. Furthermore, immunodulating therapeutic strategies, represented by cyclosporin-A discontinuation or alpha interferon treatment, may successfully reduce the Ph1+ cell population even after a full relapse.     

Induction of sister chromatid exchanges by transformation with simian virus 40.

The frequency of sister chromatid exchange (SCE) has been followed sequentially after the addition of SV40 to human diploid fibroblast cultures. The SCE frequency was nearly the same in uninfected controls and in infected cultures before they became tumor antigen positive. When cells exhibited tumor antigen, the SCE frequency increased over a wide range, and changes in chromosome number and structure were observed simultaneously. Cells with induced chromosome abnormalities without increased SCE's and the reverse present the possibility that the two phenomena have different viral mechanisms. This increase in SCE can be added to the previously demonstrated change in chromosome number and increase in chromosome breakage and rearrangement as indicators of genetic damage associated with viral transformation.     

Src-family kinases in the development and therapy of Philadelphia chromosome-positive chronic myeloid leukemia and acute lymphoblastic leukemia

The BCR-ABL kinase inhibitor imatinib has shown significant efficacy in chronic myeloid leukemia (CML) and is the standard front-line therapy for patients in chronic phase. However, a substantial number of patients are either primarily refractory or acquire resistance to imatinib. While a number of mechanisms are known to confer resistance to imatinib, increasing evidence has demonstrated a role for BCR-ABL - independent pathways. The Src-family kinases (SFKs) are one such pathway and have been implicated in imatinib resistance. Additionally, these kinases are key to the progression of CML and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The dual SFK/BCR-ABL inhibitor dasatinib is now clinically available and has markedly greater potency compared with imatinib against native BCR-ABL and the majority of imatinib-resistant BCR-ABL mutants. Therefore, this agent, as well as other dual SFK/BCR-ABL inhibitors under development, could provide added therapeutic advantages by overcoming both BCR-ABL - dependent (i.e. BCR-ABL mutations) and - independent forms of imatinib resistance and delaying transition to advanced phase disease. In this review, we discuss the preclinical and clinical evidence demonstrating the involvement of SFKs in imatinib resistance and the progression of CML and Ph+ ALL, as well as the potential role of dual SFK/BCR-ABL inhibition in the management of these diseases.     

Sister chromatid exchanges and chromosome aberrations induced by radiosensitizing agents in bone marrow cells of treated tumor-bearing mice

The frequency of sister chromatid exchanges (SCE) in vivo and chromosome aberrations and/or alterations were analyzed from the bone marrow cells of the treated dbrB tumor-bearing DBA/1J inbred mouse host. The results were compared with analogous data obtained from the bone marrow cells of untreated tumor-bearing mice for evaluation of the "indirect," i.e., somatic stress, effect on the normal host cells following triple-agent therapy intended for a mammary adenocarcinoma. Misonidazole (MIS), which is a known radiosensitizing drug, microwave hyperthermia (delta), and X-radiation (X) were used as therapeutic agents. Significant (P less than 0.05) numbers of SCE were induced in the bone marrow cells of the mice whose tumors received these triple-agent treatments (MIS + delta + X) simultaneously as compared with values of SCE per cell noted in bone marrow cells of untreated tumor-bearing control mice. The highest number of chromosome aberrations and alterations, including an increase in heteroploidy, was also noticed in the bone marrow cells of the mice whose tumors were treated simultaneously with MIS + delta + X. The triple-agent therapy on dbrB tumor also resulted in an unusually high polyploid metaphase plate in the bone marrow cell consisting of 320 chromosomes, indicating that this mode of therapy may act directly on the genetic material of the tumor-bearing host cells, inducing cytogenetic abnormalities as a side effect.     

Novel Abl kinase inhibitors in chronic myeloid leukemia in blastic phase and Philadelphia chromosome-positive acute lymphoblastic leukemia

Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia chromosome, which is associated with a balanced translocation involving chromosomes 9 and 22 to produce a fusion gene (bcr-abl) that gives rise to a constitutively activated Abl tyrosine kinase. This kinase led to the discovery of several small-molecule inhibitors, imatinib being the first and most successful of these. Resistance to imatinib results in some patients from Abl kinase point mutations. Overcoming imatinib resistance represents one of the biggest challenges facing clinicians in the modern management of CML. In this review, we discuss the current understanding of CML pathophysiology and mechanisms of imatinib resistance and how advancing this knowledge has led to the design of novel therapies in the area of blastic phase CML and Philadelphia chromosome-positive acute lymphoblastic leukemia with previous imatinib failure.     

Increased fragile sites and sister chromatid exchanges in bone marrow and peripheral blood of young cigarette smokers

Cigarette smoking is considered to be the single most important acquired cause of cancer mortality. Studies of chromosome aberrations, sister chromatid exchanges, and fragile sites in peripheral blood or bone marrow are useful methods to detect the effects of the environmental mutagens or carcinogens found in cigarette smoke. The effects of smoking on the immature cells in the bone marrow have not been studied. Here, we examine the peripheral blood and bone marrow in 18 smokers (15 females and 3 males) with a median age of 25 years (range, 21-40) and an average cigarette use corresponding to 6 pack years. In both bone marrow cells and peripheral blood lymphocytes, we were able to show a significantly increased frequency of sister chromatid exchanges in smokers with a 5 or more cigarette pack year history, but not in those who smoked less than 5 pack years. We also found a higher frequency of sister chromatid exchanges in peripheral blood lymphocytes than in bone marrow cells. In addition, the peripheral lymphocytes of smokers demonstrated (a) a significantly higher frequency of fragile sites, (b) an increased number of metaphases with extensive breakage; and (c) elevated expression of fragile sites at the cancer breakpoints 3p14.2, 11q13.3, 22q12.2, and 11p13-p14.2 and at the oncogene sites bcl 1, erb B, erb A, and sis. Our results suggest that chromosomal DNA of peripheral blood lymphocytes is sensitive to cigarette smoking. Studies of the chromosomal changes in these cells provide an index of the mutagenic damage caused by these exogenous agents in individual patients and the ability of individuals to repair that damage, and might predict susceptibility to malignant events.     

Sister chromatid exchanges and chromosomal aberrations in lymphocytes of nurses handling cytostatic agents

A cohort study of 29 nurses who constantly handled cytostatic drugs, and 29 controls matched according to sex and age, was carried out between 1983 and 1986. Cytogenetic damage was assessed by sister chromatid exchanges (SCE) and chromosomal aberrations. No significant increase in mean number of SCE was found for nurses (7.37) as compared to matched controls (7.00), whereas a significant excess of SCE (p less than 0.001) was observed for smokers (8.23) as compared to non-smokers (6.75). The number of SCE was studied in relation to the amount and nature of cytostatics handled as well as to the duration of exposure. A significant association (p less than 0.05) was found between individual mean number of SCE and the total number of drugs handled after adjustment for confounding factors. In contrast, the number of SCE was not significantly related to the nature of drugs handled or to the duration of exposure. With regard to chromosomal damage, no significant difference was observed between nurses and controls in gap, break, dicentric and translocation frequencies.     

Role of mismatch repair in the induction of chromosomal aberrations and sister chromatid exchanges in cells treated with different chemotherapeutic agents

Purpose The mismatch repair (MMR) system plays a major role in mediating the cytotoxicity and clastogenicity of agents generating O⁶-methylguanine in DNA. Loss of MMR has also been associated with tumor cell resistance to the cytotoxic effects of 6-thioguanine and cisplatin and with hypersensitivity to N-(2-chloroethyl)-N-cyclohexyl-N-nitrosourea (CCNU). The aim of the present investigation was to elucidate the role played by the MMR system in the generation of chromosomal damage in cells exposed to 6-thioguanine, cisplatin or CCNU.Methods The MMR-proficient cell lines TK6 and HCT116/3-6, and their MMR-deficient counterparts MT1 and HCT116, were treated with 6-thioguanine, cisplatin or CCNU, and analyzed for cell growth inhibition and chromosomal damage. As a control, similar experiments were performed with the methylating agent temozolomide.Results Cytotoxicity, chromosomal aberrations and sister chromatid exchanges induced by 6-thioguanine and temozolomide were significantly reduced in the MMR-deficient cell lines with respect to their MMR-proficient counterparts. In contrast, although conferring some protection against cytotoxicity, the loss of MMR did not affect cytogenetic damage induced by cisplatin. CCNU produced comparable levels of cytotoxicity, chromosomal aberrations and sister chromatid exchanges in both MMR-proficient and MMR-deficient cell lines.Conclusions The MMR system is involved in the generation of chromosomal damage in cells exposed to 6-thioguanine. The system does not play a relevant role in the generation of chromosomal damage in cells treated with CDDP and does not confer protection against the clastogenic effects of CCNU, at least in the cell lines investigated.Abbreviations BG O⁶-Benzylguanine - CCNU N-(2-Chloroethyl)-N-cyclohexyl-N-nitrosourea - CDDP cis-Diamino-di-chloro-platinum (II) - MGMT O⁶-Methylguanine-DNA methyltransferase - MMR Mismatch repair - MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - sce Sister chromatid exchanges - 6-TG 6-Thioguanine - TMZ (temozolomide) 8-Carbamoyl-3-methyl-imidazo[5,1-d]-1,2,3,5-tetrazin-4(3H)-one