Peter E. Crossen

Analysis of human lymphocyte cell cycle time in culture measured by sister chromatid differential staining.

Abstract Lymphocyte cell cycle time was measured by the BUdR-Giemsa method for demonstrating sister chromatid differential staining. All 48 h cultures showed metaphases which were in their second division. This finding indicates that the recommended culture time of between 48–54 h for the analysis of 1st division metaphases in lymphocyte cultures is too long, and that a culture time of 38–40 h would be preferable. The 48 h cultures also showed a significantly higher mitotic index than the 72 h cultures suggesting that the continuous incorporation of BUdR may have a toxic effect. The majority of 72 h cultures showed 1st, 2nd and 3rd division metaphases, but there was considerable variation among donors. There was a positive correlation between the number of 2nd division metaphases and the mitotic index.

The incidence of sister chromatid exchanges in cultured human lymphocytes

The incidence of sister chromatid exchanges (SCE) in cultured human lymphocytes from 50 normal individuals was studied using the BUdR-Giemsa technique. The mean SCE frequency per metaphase was 7.9 with a standard deviation of 1.36 and a range of 1-21. The incidence of exchanges was not influenced by the age of the donor nor did the exchange rate differ between sexes. The results of this study are compared with those of previous reports and reasons for the wide variation between results discussed.

Analysis of the frequency and distribution of sister chromatid exchanges in cultured human lymphocytes

SummaryLymphocytes from 20 normal subjects (11 male and 9 female) were examined for the frequency and location of sister chromatid exchanges (SCE) by the BrdU—Giemsa method. The mean frequency of SCE was 6.37 with little significant variation. One subject had a high number of exchanges in chromosome 1 while the remainder showed a random distribution of exchanges between chromosomes. The frequency of exchanges generally increased with chromosome length. However, chromosome 1, 2 and the B group had more exchanges than expected while the E, F and G groups had less than expected. The distribution of exchanges in chromosomes 1, 2 and the B group was non-random with a concentration of exchanges below the centromere and to a lesser extent on the distal portion of the long arm. The majority of exchanges appeared to occur at the junction between the dark and light G bands. It is suggested that the concentration of exchanges may reflect differences in BrdU incorporation along the length of the chromosome.

Sister Chromatid Exchange in Cigarette Smokers

SummaryThe incidence of sister chromatid exchanges in smokers and nonsmokers was investigated. There was no difference in the SCE rate between smokers and nonsmokers, nor was there any difference between heavy (>10 per day) and light (<10 per day) smokers.

Factors influencing sister-chromatid exchange rate in cultured human lymphocytes.

Factors influencing sister-chromatid exchange (SCE) frequency in human lymphocytes were investigated. Slides treated by the hot PO4 technique showed a lower SCE rate than those treated by the fluorescence plus Giemsa (FPG) technique. Lymphocytes cultured in McCoys 5A culture medium showed a lower SCE rate than those cultured in TC 199. Neither of the 2 batches of serum tested (Gibco batch 092 and human AB) increased the SCE rate. Cultures harvested at 48 and 72 h showed similar SCE rates. The mean SCE rate in lymphocytes from 100 subjects was 10.98 with a standard deviation of 1.71. Only 3 donors fell outside the 95% confidence level. The distribution of SCE in individual cells was judged to differ significantly from normal. Cells with increased SCE contributed to the positive skewness of the distribution. Repeat cultures from 20 subjects were studied over a 3-year period. Only 3 subjects showed significant variation in SCE in successive cultures.

Giemsa banding patterns of human chromosomes

A method of obtaining differentially stained chromosomes is described. The method based on the treatment of the chromosomes with NaOH and subsequent incubation in phosphate buffer and Giemsa staining gave distinct banding patterns in both blood and bone marrow chromosomes. The banding patterns in some instances agreed closely with those of other methods but in others they were dissimilar. A probable explanation for the differences in banding patterns is the various treatments involved in producing banded chromosomes. While repetitive DNA may play a part in band formation, other factors may also be important.

Abnormal karyotypic clones in human acute leukemia: Their nature and clinical significance

Forty‐four per cent of 110 unselected patients presenting with acute myeloid, lymphatic, or stem cell leukemia had a karyotypically abnormal, clonal cell line in the bone marrow, and about 60% of these patients also had apparently normal, diploid cells coexisting with the abnormal cells. The karyotypic changes were diverse and distinctive for each patient, but acute lymphatic leukemia tended to hyperdiploidy. The karyotypic changes showed low involvement of F chromosomes, and some excess involvement of C and G chromosomes. There was little evidence of karyotypic evolution. Where therapy induced full remission, abnormal cell lines tended to be replaced by diploid cells. The incidence of karyotypic abnormality at diagnosis was not related to patient age, sex, or type of leukemia. Significantly, the presence of karyotypic abnormality did not influence the chance of leukemic remission or patient survival, except that male patients with C chromosome deficiency survived on average three times longer than those with additional C chromosomes. This study and a review of five other major studies led to the conclusion that karyotypic abnormalities of acute leukemia have little or no etiological, clinical or hematological significance, and do not influence patient survival. It would appear that cell lines characterized by karyotypic abnormality rarely determine leukemic progression, but develop within limits set by a prescribed and individual cell environment.

Genes and chromosomes in chronic B-cell leukemia

Cytogenetic analysis of patients with chronic B-cell leukemia (B-CLL) indicates that 50% have chromosome abnormalities, while fluorescence in situ hybridization (FISH) and molecular techniques reveal an even higher incidence. Trisomy 12 and deletions or translocation of chromosome 13q14 are the most common abnormalities, but in neither case has the gene or genes involved in the abnormalities been identified. Combined FISH and immunophenotyping studies suggest that both abnormalities are secondary events in B-CLL. Other recurring chromosome abnormalities include 6q-, 11q- and 12p-, but the genes involved in these abnormalities have not been identified. Involvement of the BCL1, BCL2, and BCL3 genes has been reported, but the numbers are low and the cases tend to be atypical. Trisomy 12 in association with complex karyotypic abnormalities is associated with a poor prognosis, and FISH studies show a strong correlation between trisomy 12, atypical morphology, and advanced disease. Ten to 15% of patients have mutations of p53 which is associated with advanced disease, resistance to treatment, and poor survival.

Cytogenetic and molecular changes in chronic B-cell leukemia

Cytogenetic studies using B-cell mitogens indicate that approximately 50% of patients with chronic B-cell leukemia (CLL) have chromosome abnormalities. The most common abnormality is an additional chromosome 12, either as the sole abnormality or in conjunction with other abnormalities such as 14q+, 6q-, and 11q-. In two instances, the 14q+ is a result of a translocation from either chromosome 11, t(11;14), or chromosome 19, t(14;19). These two translocations led to the identification of the bcl-1 and bcl-3 genes located on chromosomes 11 and 19, respectively. Very few instances of oncogene activation have been described and it does not seem to be an important mechanism in the pathogenesis of CLL. Further cytogenetic and molecular studies may provide clues for the identification of the genes involved in CLL.

Cell cycle analysis using bromodeoxyuridine: Comparison of methods for analysis of total cell transit time

Results are presented supporting the study of cellular proliferation utilizing 5-bromodeoxyuridine (BrdU) incorporation followed by sister chromatid differential staining. In order to determine the relative accuracy of this method in estimating total cell transit time (TC), we utilized a thermosensitive rat embryonic cell line to compare measurement of TC based on the percent differentially labeled (PDLM) technique, with cell cycle measurements using [3H]-thymidine [( 3H]-TdR) incorporation and the percent labeled mitoses (PLM) technique. Results of PLM and PDLM analysis were shown to be highly concordant, indicating the utility of the BrdU method for analysis of Tc. Results are presented suggesting the general application of the PDLM method for estimations of TC in cultures of human tumors.