Lisa Staiano-Coico

Increased chromosomal instability in lymphocytes from elderly humans

Lymphocytes from young and old donors were incubated with PHA for 96 h and exposed to [3H]Tdr during the last 24 h of culture. Comparable amounts of [3H]Tdr were incorporated into chromosomes of old and young lymphocytes as measured by autoradiography of metaphase chromosomes. However, chromosomal damage and cell-cycle arrest were far greater in lymphocytes from old as compared to young humans. The frequency of chromosome breaks, fragments, exchange figures and dicentric chromosomes induced by [3H]Tdr was greater in cultures from old than in cultures from young humans. Lymphocytes from old donors exposed to 20 microM BrdU during the last 24 h of culture showed significantly more sister-chromatid exchanges than did lymphocytes from young donors. These data suggest that chromosomes in lymphocytes from old donors express more damage after exposure to [3H]Tdr or BrdU than do chromosomes in lymphocytes from young donors.

Monitoring intravesical bacillus Calmette-Guerin treatment of bladder carcinoma with flow cytometry.

Flow cytometry of bladder irrigation specimens was studied in 22 patients with low stage bladder carcinoma who were treated by transurethral resection of visible tumor followed in 3 to 5 weeks by a course of intravesical bacillus Calmette-Guerin. The most informative examinations were just before the first bacillus Calmette-Guerin treatment, 6 weeks after completing a 6-week course of treatment (3 months) and at 9 months. Of the patients 10 had recurrent tumors after therapy; recurrence was anticipated correctly by flow cytometry at the 12-week followup examination in 6 of the 10 patients and suspected in another. Of 12 patients who remained clinically free of disease for a minimum of 15 months after bacillus Calmette-Guerin therapy flow cytometry identified correctly 7 at 12 weeks, while 1 had a partial response and the remaining 4 reverted to a negative status at 9 months. Of interest, only 4 of the 22 patients were free of disease by flow cytometry at the start of bacillus Calmette-Guerin treatment despite attempted ablation of the tumor by transurethral resection, suggesting that intravesical administration of bacillus Calmette-Guerin destroys existing carcinoma in situ in some cases.

Presumptive downstaging from preoperative irradiation for bladder cancer as determined by flow cytometry: Preliminary report

Presumptive tumor downstaging was evaluated in 28 patients with grade II or III, solid, muscle-infiltrating bladder cancer (clinical category T3) treated by integrated irradiation (2000 rad to the whole pelvis in 5 days) and cystectomy (1-14 days later) by comparing the results of flow cytometry (FCM) on barbotage specimens obtained before and after irradiation (at the time of cystectomy) and the results of pretreatment clinical stage (T category) and post cystectomy pathological stage (P category). The patients were divided into three groups: (1) P greater than T, (2) P = T, and (3) P less than T. All of the patients in this study had positive FCM specimens with an aneuploid stemline in the pre-irradiation specimen. A complete radiation response (CRR) was defined by FCM as disappearance of the aneuploid stem cell line. Of the 5 patients in the P less than T group, 4 showed a CRR; of 20 patients in the P = T group, 8 showed a CRR; of the 3 patients in the P greater than T group, none showed a CRR. The proportion of patients in the various T/P groups is consistent with that previously observed in patients receiving integrated irradiation (2000 rad in 5 days) and cystectomy (1-14 days later). The overall downstaging response of 43%, as determined by FCM, correlates well with the pathological downstaging rates of 40%-68% reported by others following high dose (4000-5000 rad) integrated irradiation cystectomy regimens; however, it is more than the 27% rate reported with the low dose short course (2000 rad in 5 days) regimen. The correlation of the FCM findings with clinico-pathological downstaging is consistent with the possibility that FCM may be useful in identifying a favorable radiation response.

P53 content in relation to cell growth and proliferation in murine L1210 leukemia and normal lymphocytes

The protein p53 has been reported to be associated with cell transformation and/or proliferation. Using p53 monoclonal antibodies we estimated by flow cytometry the relative content of this protein in individual L1210 leukemic cells from exponentially growing and plateau-phase cultures and compared it with that in normal thymocytes of parental DBA/2 mice and in mitogen-stimulation and nonstimulated human lymphocytes. Simultaneous differential staining of p53 vs DNA and p53 vs RNA, followed by bivariate analysis, made it possible to estimate p53 with respect to cell position in the cell cycle and correlate it with RNA (predominantly rRNA) content. The data show that in exponentially growing L1210 cells p53 is being progressively accumulated during the G1, S and G2 phases and that the content of p53 and RNA are highly correlated. In plateau L1210 cultures most cells are arrested in G1, some cells, however, still continue to progress through S and G2. In these cultures the p53 content of all cells, regardless of the phase of the cell cycle, is diminished and the decrease in p53 is more pronounced than that of RNA or total protein content. The normal thymocytes as well as the stimulated lymphocytes show bimodal distribution with respect to p53 expression, compatible with the assumption that the cycling cells have increased expression of this protein related to the G0 cells. Some cycling cells, however, have minimal p53. The quantitative p53 immunofluorescence data were confirmed by the immunoprecipitation and gel electrophoresis. The results suggest that expression of p53 in leukemic and normal cells is more correlated with cell growth than with entrance to the cell cycle or progression through particular phases of the cycle.

Binding of anti-Z-DNA antibodies in quiescent and activated lymphocytes: relationship to cell cycle progression and chromatin changes.

Although regions of DNA reacting with anti-Z-DNA antibodies have been identified in the polytene chromosomes of Drosophila spp. and the metaphase chromosomes from a number of different mammalian species, the biological role of this DNA is unknown. Flow cytometry was used in the present studies to quantitate the binding of anti-Z-DNA antibodies in quiescent and activated human peripheral blood lymphocytes; the antibody binding was then correlated with cell cycle phase. The data show that quiescent (G0 or G1Q) lymphocytes are heterogeneous with respect to their reaction with anti-Z-DNA antibodies. The transition from quiescence (G1Q) into the cell cycle (G1), which involves decondensation of chromatin, did not result in any significant change in binding of these antibodies. In contrast, progression of cells from G1 through S and G2 is correlated with a 27% decrease in anti-Z-DNA antibody reactivity relative to total DNA content. No significant change was observed during the transition from G2 to mitosis (M).