Premature Chromosome Condensation Technique Research Articles

Detection of leukemic clone maturation in vivo by premature chromosome condensation

The purpose of this study was to determine the feasibility of using the technique of premature chromosome condensation to detect the in vivo maturation of abnormal elements in patients with chronic myelogenous leukemia (CML), myelodysplastic syndrome, and acute leukemia. Patients were chosen for study if there were a clinical suggestion of in vivo maturation and a leukemic clone exhibiting a distinguishable karyotypic abnormality. Mature peripheral blood granulocytes were enriched by two- step Ficoll-Hypaque gradient sedimentation and fused with mitotic Chinese hamster ovary cells to induce the formation of prematurely condensed chromosomes (PCC). These PCC were then analyzed for chromosome number per cell (in the case of patients with a numerical abnormality) or by G-banding (in the case of specific translocations). Of 13 patients chosen for study, 12 showed karyotypic evidence for maturation of the abnormal elements in vivo. Maturation was observed in a number of clinical situations including before treatment in benign CML and myelodysplasia, after low-dose and high-dose chemotherapy in myelodysplasia and acute myelogenous leukemia (AML), and in remission. These results suggest that the technique of premature chromosome condensation can be a powerful tool in better understanding the biology of disease and mode of response to therapy in vivo in patients with leukemia and preleukemic syndromes, especially during treatment with agents thought to induce maturation of the leukemic elements.

Improvements in the premature chromosome condensation technique for cytogenetic analysis

In this paper we report a combination of procedures that serve to improve the usefulness of the technique of premature chromosome condensation in cytogenetic investigations. The mitotic inducer population was preincubated in high concentrations of BrdU, and the duration of fusion was increased to yield more discrete prematurely condensed chromosomes (PCC). After fusion, chromosome preparations were treated with a combination of G- or C-banding techniques and differential staining techniques. This combination of procedures allowed unequivocal distinction between the PCC and mitotic inducer chromosomes and yielded banded G1 and G2 PCC suitable for routine cytogenetic investigations.

Direct analysis of the clastogenic effect of formaldehyde in unstimulated human lymphocytes by means of the premature chromosome condensation technique

The cytogenetic effect of formaldehyde (FA) on unstimulated human lymphocytes was studied by means of conventional chromosome analysis and the premature chromosome condensation (PCC) technique. In first post-treatment metaphases no significantly increased yields of chromosomal changes could be observed. The analysis of PCCs, however, showed high yields of chromosome fragments. Bleomycin (BLM) used as positive control was also highly clastogenic in PCCs and resulted in significantly increased yields of chromosome-type aberrations. As recently argued, a premitotic selection against heavily damaged cells could be an explanation for the discrepancy between the chromosome findings in metaphase and PCC analysis after FA treatment. In addition, a differential effectiveness may exist in unstimulated lymphocytes to convert multiple fragmentation into chromatid- or chromosome-type aberrations through S-phase-dependent or S-phase-independent mechanisms.

37 Direct solid tumor cytogenetics using the technique of premature chromosome condensation: Walter N. Hittelman, Phylisha Agbor, and Iskra Petkovic. University of Texas M. D. Anderson Hospital, Houston, Texas 77030

37 Direct solid tumor cytogenetics using the technique of premature chromosome condensation: Walter N. Hittelman, Phylisha Agbor, and Iskra Petkovic. University of Texas M. D. Anderson Hospital, Houston, Texas 77030

Premature chromosome condensation--studies on human metastatic carcinoma cells.

Utilizing the phenomenon of premature chromosome condensation (PCC) studies were carried out on interphase chromatin of metastatic cells from 52 cancerous effusions obtained from 45 patients presenting with various solid carcinomas. A highly individual pattern of distribution of the various interphase stages was detected, reflecting the heterogeneity of human solid tumors in an advanced stage. Nevertheless a variety of clinical, biologic, and technical factors were examined for their possible influence on these PCC patterns. The duration in culture was one of the influencing factors, as were the time lapse between the first diagnosis and the sampling of the respective effusion, or the nature of cytostatic therapy. Cytogenetic equivalents of gene amplification, as represented by "double minutes", could be found in the prematurely interphase chromatin of 35 of the 52 effusions. G1-PCC proved to be most reliable with regard to screening of double minutes. In addition, an adequate quality of Giemsa banding was achieved in PCC of 21 out of 24 effusions yielding a sufficient number of well-spread PCC. In six of these 21 cases PCC was superior to metaphase analysis in obtaining karyotypes, while the same was true for 14 of the 52 effusions screened for double minutes. Thus the PCC technique was shown to be an indispensable additional source of cytogenetic information in cells of human solid tumors.

Radiation-Induced Cytogenetic Damage in Relation to Changes in Interphase Chromosome Conformation

The premature chromosome condensation (PCC) technique was used to study several factors that determine the yield of chromosome fragments as observed in interphase cells after irradiation. In addition to absorbed dose and the extent of chromosome condensation at the time of irradiation, changes in chromosome conformation as cells progressed through the cell cycle after irradiation affected dramatically the yield of chromosome fragments observed. As a test of the effect of chromosome decondensation, irradiated metaphase Chinese hamster ovary (CHO) cells were allowed to divide, and the prematurely condensed chromosomes in the daughter cells were analyzed in their G1 phase. The yield of chromosome fragments increased as the daughter cells progressed toward S phase and chromosome decondensation occurred. When early G1 CHO cells were irradiated and analyzed at later times in G1 phase, an increase in chromosome fragmentation again followed the gradual increase in chromosome decondensation. As a test of the effect of chromosome condensation, G0 human lymphocytes were irradiated and analyzed at various times after fusion with mitotic CHO cells, i.e., as condensation proceeded. The yield of fragments observed was directly related to the amount of chromosome condensation allowed to take place after irradiation and inversely related to the extent of chromosome condensation at the time of irradiation. It can be concluded that changes in chromosome conformation interfered with rejoining processes. In contrast, resting chromosomes (as in G0 lymphocytes irradiated before fusion) showed efficient rejoining. These results support the hypothesis that cytogenetic lesions become observable chromosome breaks when chromosome condensation or decondensation occurs during the cell cycle.

A cytogenetic investigation of DNA rereplication after hydroxyurea treatment: implications for gene amplification.

Although the mechanisms leading to gene amplification are poorly understood, it has recently been proposed that the initial event of amplification is the rereplication of a variable, but relatively large, amount of the genome within a single cell cycle. We sought evidence for rereplication of DNA as a basis for gene amplification through two cytogenetic techniques: differential staining for sister-chromatid exchange analysis and premature chromosome condensation. Synchronized Chinese hamster ovary cells were incubated continuously with bromodeoxyuridine and treated with hydroxyurea (HU) when cells were approximately 2 h into the S phase. After 6 h exposure to HU, the drug was removed and at 3 h intervals thereafter metaphase cells were collected and the chromosomes were stained by the fluorescence-plus-Giemsa procedure. No staining patterns consistent with rereplication of DNA were observed. Since HU causes cytogenetic damage, the premature chromosome condensation technique was used to determine the kinetics of chromosome damage after removal of HU. Extensive G2 chromosome damage within 1 h after removal of HU from the medium was found, although cesium chloride gradient analysis showed that there was no rereplication of DNA during this time. Contrary to a previous report, these results provide no evidence that incubation of cells with HU during S phase induces rereplication of DNA within a single cell cycle. The results observed are consistent with the hypothesis that drug-induced aberrations and the subsequent abnormal segregation of chromosomal fragments are the first steps in the process that leads to gene amplification in drug-treated mammalian cells.

Cytosine arabinoside is a potent clastogen and does not affect the repair of X-ray-induced chromosome fragments in unstimulated human lymphocytes

The metabolic inhibitor of DNA synthesis cytosine arabinoside (ara-C) is known to induce chromosome aberrations in human lymphocytes. It has been recently argued, however, that there is no unequivocal evidence that ara-C can damage chromosomes directly. Therefore, the effect of ara-C on unstimulated human lymphocytes was examined directly by means of the premature chromosome condensation technique. I In about 50% of the cells, ara-C effectively induced chromosome fragments, which did not show rejoining even after the chemical was washed out. These results suggest that a possible selection against damaged cells in their progress to mitosis could result in the low yields of ara-C-induced chromosome aberrations reported in the literature. The effect of ara-C on the repair of radiation-induced chromosome aberrations was also examined. Ara-C did not affect the rejoining of the chromosome fragments induced in unstimulated human lymphocytes by 6 Gy of X-rays.

Direct analysis of radiation-induced chromosome fragments and rings in unstimulated human peripheral blood lymphocytes by means of the premature chromosome condensation technique

Development of the procedure to stimulate peripheral blood lymphocytes has greatly facilitated the understanding of chromosome aberration formation and repair mechanisms in human cells. Yet, because radiation induces far more initial chromosome breaks than are observed as aberrations in metaphase, it has not been possible to examine the kinetics of primary chromosome breakage and rejoining with this procedure. An improved method to induce premature chromosome condensation is unstimulated lymphocytes has been used to study primary chromosome breakage, rejoining, and ring formation at various times after irradiation with up to 800 rad of X-rays. The dose-response relations for chromosome fragments analyzed immediately or 1, 2, or 24 h after exposure were found to be linear. Rapid rejoining of chromosome fragments, which takes place in the first 3 h after X-ray exposure, was not correlated with a simultaneous increase in the formation of rings. The yield of rings per cell scored 24 h after irradiation, however, increased significantly and fit a linear quadratic equation. Both chromosome fragment rejoining and ring formation were completed about 6 h after irradiation. The frequency distributions of rings among cells followed a Poisson distribution, whereas chromosome fragments were overdispersed.

Premature chromosome condensation studies in human leukemia: 5. Prediction of early relapse

Previous reports have suggested that the technique of premature chromosome condensation (PCC) is useful for predicting relapse in patients with acute leukemia. However, these studies involved patients had been in complete remission (CR) for various periods of time and had heterogeneous expectations for relapse. The purpose of this study was to further determine the value of PCC in predicting relapse by examining the PCC characteristics of bone marrow specimens from patients with acute leukemia on a common therapeutic regimen after similar periods in CR. The remission durations after the PCC determinations were compared between patients with high or low proliferative potential indices (PPI, or the fraction of G1 cells in late G1 phase). Of 60 patients studied between two and eight weeks after achieving CR, 14 of the 16 patients exhibiting high PPI values (greater than or equal to 35) have relapsed. The mean time from PCC measurement to relapse was 23 weeks. In contrast, only 19 of the 44 patients exhibiting low PPI values have relapsed, with an estimated mean time to relapse of 68+ weeks. Likewise, of 38 patients studied between nine and 15 weeks of CR, nine of the ten patients exhibiting high PPI values have relapsed (mean time to relapse, 23 weeks), while only 16 of 28 patients with low PPI values have relapsed (estimated mean time to relapse, 54+ weeks). The predictive value of the PCC technique was found to be independent of other prognostic factors for the duration of CR, and it identified those patients within the poor prognostic category with a high likelihood of imminent relapse. While similar trends were observed at later time intervals in CR, the differences in relapse rate between patients with high or low PPI values is not significant. These results confirm the usefulness of the PCC technique in predicting relapse in acute leukemia and could aid in the identification of patients who might benefit by an alteration of therapeutic strategy.

Premature Chromosome Condensation Studies in Human Leukemia 5. Prediction of Early Relapse

Previous reports have suggested that the technique of premature chromosome condensation (PCC) is useful for predicting relapse in patients with acute leukemia. However, these studies involved patients had been in complete remission (CR) for various periods of time and had heterogeneous expectations for relapse. The purpose of this study was to further determine the value of PCC in predicting relapse by examining the PCC characteristics of bone marrow specimens from patients with acute leukemia on a common therapeutic regimen after similar periods in CR. The remission durations after the PCC determinations were compared between patients with high or low proliferative potential indices (PPI, or the fraction of G1 cells in late G1 phase). Of 60 patients studied between two and eight weeks after achieving CR, 14 of the 16 patients exhibiting high PPI values (greater than or equal to 35) have relapsed. The mean time from PCC measurement to relapse was 23 weeks. In contrast, only 19 of the 44 patients exhibiting low PPI values have relapsed, with an estimated mean time to relapse of 68+ weeks. Likewise, of 38 patients studied between nine and 15 weeks of CR, nine of the ten patients exhibiting high PPI values have relapsed (mean time to relapse, 23 weeks), while only 16 of 28 patients with low PPI values have relapsed (estimated mean time to relapse, 54+ weeks). The predictive value of the PCC technique was found to be independent of other prognostic factors for the duration of CR, and it identified those patients within the poor prognostic category with a high likelihood of imminent relapse. While similar trends were observed at later time intervals in CR, the differences in relapse rate between patients with high or low PPI values is not significant. These results confirm the usefulness of the PCC technique in predicting relapse in acute leukemia and could aid in the identification of patients who might benefit by an alteration of therapeutic strategy.

The Use of Peripheral Blood Mononuclear Cell Prematurely Condensed Chromosomes for Biological Dosimetry

Even though the premature chromosome condensation (PCC) technique has been shown to be a powerful cytogenetic tool, it has not yet been applied for biological dosimetry purposes. An improved and simple methodology for polyethylene glycol-mediated cell fusion and PCC induction has given the opportunity to explore the use of peripheral blood mononuclear cell prematurely condensed chromosomes as a biological dosimeter of absorbed radiation dose. This new approach offers several advantages, and it is presented as an alternative to the conventional cytogenetic technique. First, the chromosomal damage may be visualized in about 2 hr from the moment a blood sample is available. Consequently, the chances for interphase cell death or chromosomal repair are decreased. Second, the amount of blood required is only 0.5 ml. Third, the PCC analysis of about 100 mononuclear blood cells shows the radiation damage even at low doses, whereas hundreds of lymphocyte metaphases must be analyzed when conventional cytogenetic techniques are applied. Fourth, the linear dose-response relationships obtained may be easily standardized. The methodology used diminishes the possibility of differential results from one laboratory to another by avoiding the variables introduced by the stimulation and culture of lymphocytes required by conventional cytogenetic techniques. Finally, the kinetics of lymphocyte chromosome aberration recovery can be directly examined. This approach provides us, therefore, with a direct and more sensitive method for the estimation of radiation injury and repair.

Cytogenetic analysis of urologic malignancies: study of tumor colony forming cells and premature chromosome condensation.

We have utilized a human tumor clonogenic assay to grow, and subsequently cytogenetically analyze, tumor colony forming cells from human urologic malignancies. Results following chromosome banding analysis are presented from 4 cases of transitional cell carcinoma and 1 case of renal cell carcinoma. Preliminary evidence suggests a possible association between the loss of chromosome 8 and progression or recurrence of transitional cell carcinoma. Additionally, we have utilized the technique of premature chromosome condensation to identify the interphase chromatin profile of urothelial cells obtained by cystoscopy from 8 patients with transitional cell carcinoma and compared these results to urothelial cells obtained from 7 control patients. This study demonstrates that cells obtained from urologic cancers contain a high proportion of cells in late G1, while normal urothelial cells are usually found in the early G1 phase of the cell cycle. Statistical correlation of premature chromosome condensation analysis suggests this method may be a useful adjunct to routine histopathology in discriminating between normal and cancerous urothelium.

A Comparison of the DNA and Chromosome Repair Kinetics after γ Irradiation

The kinetics of repair at the chromosome and DNA levels were compared after ..gamma.. irradiation of Chinese hamster ovary cells (CHO). Induction and repair of DNA damage were measured by the alkaline and neutral elution techniques, while chromosome damage and repair were determined by the technique of premature chromosome condensation. During and after ..gamma.. irradiation, significant DNA repair occurred within 2 min. This fast repair could be inhibited by EDTA and pyrophosphate and probably reflected polynucleotide ligase activity. A slower component of DNA repair was detected between 15 and 60 min after irradiation, by which time most of the DNA had been repaired. In contrast, chromosome repair was not detectable until 45 min after irradiation, and nearly half of the chromatid breaks were repaired by 60 min. Cycloheximide, an inhibitor of protein synthesis, prevented chromosome break repair, yet had no effect on the immediate formation of chromatid exchanges or DNA repair. These results suggest the following: (1) the rapidly repairing DNA lesions are not important in the repair of chromosomes; (2) chromosome damage involves only a minority of the DNA lesions measured by alkaline and neutral DNA elution; and (3) chromosome repair may involve more than simply the repair ofmore » damaged DNA that can be detected by the alkaline and neutral elution assays.« less

Premature chromosome condensation studies in human leukemia: 4. Characterization of albumin density fractionations of bone marrow at presentation, remission, and relapse

Previous studies have suggested that the technique of premature chromosome condensation (PCC) is useful in the study of human leukemia, both as a predictive tool for course of disease and as a probe to better understand the biology of the disease process. The purpose of this study was to determine how the various subcomponents of bone marrow populations contribute to the overall PCC pattern. Thirty bone marrow aspirations from persons at various stages of disease (2 normal, 11 untreated, 12 in remission, and 5 n relapse) were fractionated according to density in albumin gradients, and the various fractions were characterized by determining the proliferative potential index (PPI, or the fraction of G1 cells in late G1) using the PCC technique. In general, normal bone marrow and marrow from patients In remission showed lower PPI values throughout the gradients, with the most dense and most mature cells yielding the lowest PPI values in the gradient. In contrast, bone marrow from newly presenting patients and from patients in relapse showed higher PPI values, with the highest PPI values found In the most dense fractions containing the most mature cells. Thus, residual mature cells from patients with active disease exhibit the malignant characteristic of accumulating in late G1 phase. It is postulated that these morphologically mature cells from patients with active disease either represent matured cells of malignant origin or represent residual normal cells pushed beyond the restriction point in early G1 phase by a putative tumor growth factor synthesized by the leukemic cell population.

Premature Chromosome Condensation Studies in Human Leukemia: 4. Characterization of Albumin Density Fractionations of Bone Marrow at Presentation, Remission, and Relapse

AbstractPrevious studies have suggested that the technique of premature chromosome condensation (PCC) is useful in the study of human leukemia, both as a predictive tool for course of disease and as a probe to better understand the biology of the disease process. The purpose of this study was to determine how the various subcomponents of bone marrow populations contribute to the overall PCC pattern. Thirty bone marrow aspirations from persons at various stages of disease (2 normal, 11 untreated, 12 in remission, and 5 in relapse) were fractionated according to density in albumin gradients, and the various fractions were characterized by determining the proliferative potential index (PPI, or the fraction of G1 cells in late G1) using the PCC technique. In general, normal bone marrow and marrow from patients in remission showed lower PPI values throughout the gradients, with the most dense and most mature cells yielding the lowest PPI values in the gradient. In contrast, bone marrow from newly presenting patients and from patients in relapse showed higher PPI values, with the highest PPI values found in the most dense fractions containing the most mature cells. Thus, residual mature cells from patients with active disease exhibit the malignant characteristic of accumulating in late G1 phase. It is postulated that these morphologically mature cells from patients with active disease either represent matured cells of malignant origin or represent residual normal cells pushed beyond the restriction point in early G1 phase by a putative tumor growth factor synthesized by the leukemic cell population.

Isolation by a replica-plating technique of Chinese hamster temperature-sensitive cell cycle mutants.

Isolation of a wide variety of temperature-sensitive (ts) cell cycle mutants in mammalian cells has previously proved to be a very difficult task. The various procedures used for the isolation of such mutants included a mutant enrichment step based on exposure of the cells to the restrictive temperatures in order to kill the growing wild-type cells with agents that kill DNA-synthesizing cells. Hence, these methods favored the isolation of ts mutants that do not lose viability rapidly at the restrictive temperatures. We have treated cells of the Chinese hamster established cell line E36 with the mutagen ethyl-methane-sulfonate (EMS) and used a replicating technique that we developed to screen the ts mutants for growth. This technique enabled us to recover all its mutants for growth including the ts cell cycle mutants. Screening of the ts cell cycle mutants among the ts mutants for growth was performed by the flow microfluorimetry technique and the premature chromosome condensation technique. Our results show that 1.3% of the survivors of the mutagenic treatment are ts mutants for growth. Six of 84 ts mutants analyzed were found to be ts cell cycle mutants. They include ts mutants arrested in phases G1, S, and G2. Many of the ts mutants for growth including the ts cell cycle mutants arrested in S and G2 lose viability very fast when incubated at the restrictive temperature. As a consequence they could not have been isolated by any method that includes a mutant enrichment step based on the exposure of the cells to the restrictive temperature.

Predicting relapse of human leukemia by means of premature chromosome condensation.

We attempted to determine whether the technique of premature chromosome condensation (PCC) could be used to predict relapse in patients with acute leukemia in complete remission. Nineteen patients received chemotherapy and were studied serially with the PCC technique. Of the 19 patients in this pilot study, 14 relapsed, and the PCC technique predicted relapse in 11 of these 14. The median time from an elevation in the fraction of G1 cells in the late G1 phase (a high proliferative-potential index) to the development of clinical evidence of relapse was 3.5 months. On the other hand, the median time from a low proliferative-potential index to relapse or to the end of the study was eight months. The incidense of false-positive or false-negative measurements was low. Two patients had high index values just before chemotherapy, and remission was estimated to be prolonged by 2.5 and six months by our therapeutic protocol. These results suggest that the PCC technique is useful and accurate in the early prediction of relapse in human leukemia.

Premature Chromosome Condensation Studies in Human Leukemia. 2. Proliferative Potential Changes After Induction Therapy for AML Patients

Twenty-two patients with acute myelogenous leukemia (AML) were studied serially during remission induction therapy using the technique of premature chromosome condensation (PCC). Mononuclear cells from bone marrow aspirations were fused with mitotic Chinese hamster ovary cells, and the PCC were scored for fraction of cells in G1, S, and G2. The proliferative potential index PPI, or fraction of the G1 cells in late G1) and degree of chromosome damage were also determined. These results were compared to the changes in bone marrow morphology and blast counts during therapy and correlated with the clinical outcome. Patients who responded to chemotherapy generally exhibited an initial drop in PPI concommitant with chromosome damage. This was followed by a rise in the PPI and fraction of S-phase cells prior to clinical evidence of bone marrow regeneration. As the bone marrow and blood continued to regenerate to normal levels, the PPI value decreased to intermediate levels (less than 35%). In contrast, patients who did not achieve complete remission were observed to exhibit no detectable PCC changes or continued high PPI values after regeneration. High blast counts in the marrow during the regenerative phase accompanied by low PPI values were correlated with a favorable prognosis, since all patients with such a pattern achieved complete remission in that course or the subsequent course of therapy. The PCC technique, therefore, is useful in monitoring response to remission induction therapy in AML patients and can complement morphological and cytogenetic parameters. This ability might be especially useful during the regenerative period when normal and leukemic regeneration is difficult to distinguish by classical techniques.

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were pbserved not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesion that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.