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M R Melamed

Publications and source records attributed to M R Melamed.

At least 37 records · Page 2Linked to original sources
PubMed1997

Laser scanning cytometric analysis of cyclin B1 in primary human malignancies.

Cyclins are key components of the cell cycle progression machinery. They activate their partner-dependent kinases (CDKs) and target them to respective substrate proteins within the cell. CDK-mediated phosphorylation of specific sets of proteins drives the cell through particular phases or checkpoints of the cell cycle. During unperturbed growth of normal cells, the timing of expression of several cyclins is discontinuous, occurring at discrete and well-defined periods of the cell cycle. Immunocytochemical detection of cyclins in relation to cell cycle position (DNA content) by multiparameter flow cytometric techniques has provided a new approach to cell cycle studies. This approach, like no other method, can be used to detect the "unscheduled" expression of cyclins, namely, the presentation of G1 cyclins by cells in G2/M and of G2/M cyclins by G1 cells, without the need for cell synchronization. By use of multiparameter flow cytometric and laser scanning cytometric analysis, we correlated the expression of cyclin B1 with cell cycle position in normal lymphocytes stimulated to proliferate by the mitogen phytohemagglutinin and in 28 primary human tumors of different organ and type. Eighteen of the 28 tumors expressed the cyclin B1 in more than 5% of cells (B1 positive), and the rest showed cyclin expression from 2.1 to 5% (B1 negative). In normal lymphocytes, the expression of cyclin B1 was restricted to very late S and G2 + M phases of the cell cycle. In 15 of 18 primary tumors studied, the expression of cyclin B1 was "unscheduled" (unrestricted to particular phases of the cycle). The data suggest that the "unscheduled" expression of cyclin B1 might be a common defect in neoplasia.

Adrenal Gland Neoplasms↗
PubMed1996

Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color.

Selective DNA strand break induction by photolysis (SBIP) at sites that contain incorporated halogenated nucleotides has been recently proposed as a means of analyzing DNA replication and detecting proliferating cells. The presence of numerous in situ DNA strand breaks is also an inherent feature of apoptotic cells. The aim of the present study was to differentially label DNA strand breaks in apoptotic cells vs photolysis-induced breaks in BrdUrd incorporating cells. This would allow one, by multicolor staining, to identify these respective cells in the same sample preparation. Toward this end, exponentially growing HL-60 cells were pulse labeled with BrdUrd and then were subjected to hyperthermia or treated with DNA topoisomerase I inhibitor camptothecin to induce apoptosis. DNA strand breaks in apoptotic cells were first labeled directly with fluorochrome-conjugated dUTP or dCTP, followed by dideoxynucleotide (to terminate chain elongation), in a reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The cells were subsequently exposed to UV light to photolyze DNA containing the incorporated BrdUrd. The photolysis-induced DNA strand breaks were, in turn, labeled with digoxygenin- or biotin-conjugated dUTP followed by digoxygenin antibody or avidin, respectively, conjugated with fluorochrome of another color. Alternatively, DNA strand breaks were labeled with BrdUTP which was then detected by FITC-conjugated anti-BrdUrd MoAb. Following counterstaining of cellular DNA with a fluorochrome of a third color it was possible to identify apoptotic cells, cells incorporating BrdUrd, and cells having no DNA strand breaks. Cell fluorescence was measured either by flow cytometry or with the microscope-based laser scanning cytometer. The SBIP approach also offers a possibility to study a colocalization of the immunocytochemically detectable cell constituents at the DNA replication points by microscopy. Using this approach the presence of the proliferating cell nuclear antigen at the DNA replication sites was revealed in MCF-7 breast carcinoma cells.

Antibodies, Monoclonal↗
PubMed1996

Third-color CD45 staining of paraformaldehyde-fixed peripheral blood lymphocytes.

Third-color CD45 gating is a useful procedure in various clinical applications (Borowitz et al.: Am J Clin Pathol 100:534-540, 1993; Steltzer et al.: Ann NY Acad Sci 677:267-280, 1993) allowing for the selective immunophenotyping of abnormal cell populations. In laboratories where dual-color protocols are in place, the need for CD45 gating may not become evident until after cells have been exposed to fixatives. This brief study demonstrates that third-color CD45 staining and gating is possible with paraformaldehyde fixed lymphocytes, eliminating the need to restain fresh cells with the entire antibody panel, and with a significant saving of time and money. Gates must be modified from those obtained by fresh cell staining in order to include both bright and dim CD45 populations. In a standard protocol, third-color CD45 staining intensity was decreased 10-fold post-fixation with paraformaldehyde except, unexpectedly, for CD8+ cells. The latter phenomenon could be explained if the spatial arrangement of CD45 and CD8 is such that the CD45 epitope is protected from cross-linking by paraformaldehyde fixation in CD8+ cells, but not others.

CD8 Antigens↗
PubMed1996

Aberrations of chromosomes 9 and 17 in bilharzial bladder cancer as detected by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH), using repetitive alpha satellite probes to chromosomes 9 and 17, was performed on 27 paraffinembedded bladder specimens from 18 Egyptian patients affected with bilharzial carcinoma. Fluorescence in situ hybridization results in the carcinomas and benign mucosa of patients with schistosomiasis were compared with flow cytometric DNA ploidy and cell cycle assays. These results demonstrate the feasibility of this technique for studies of these archival specimens, and suggest early onset of chromosomal aberrations in histologically benign mucosa of schistosomal cystitis with progression during the development of bladder cancer. On the basis of these data, monosomy 9 may be an early chromosomal change in urothelium of the bilharzia-infested bladder, and a predictor of incipient carcinoma in patients with bilharzial cystitis.

Carcinoma↗
PubMed1996

Mucinous (colloid) carcinoma of urinary bladder following long-term cyclophosphamide therapy for Waldenstrom's macroglobulinemia.

Carcinoma of the urinary bladder is a known complication of cyclophosphamide therapy. Almost all such cases have been transitional cell carcinomas. We report here the second example of an adenocarcinoma of bladder and the first purely mucinous (colloid) carcinoma of urinary bladder developing after long-term cyclophosphamide therapy. The patient, a 77-year-old woman, had been treated for Waldenstrom's macroglobulinemia for at least 24 years, during which time treatment for this disease varied from 50 to 100 mg per day. The disease terminated in acute myelogenous leukemia, and she died of severe disseminated intravascular coagulopathy associated with hypermacroglobulinemia. The mucinous (colloid) carcinoma of the urinary bladder was an incidental finding at autopsy.

Adenocarcinoma, Mucinous↗
PubMed1996

Laser scanning cytometer (LSC) analysis of fraction of labelled mitoses (FLM).

In this report we describe the successful application of a novel microscope-based multiparameter laser scanning cytometer (LSC) to measure duration of different phases of cell cycle in HL-60 human leukaemic cell lines by the fraction of labelled mitoses (FLM) method. Exponentially growing cells were harvested after various time intervals following pulse-labelling with 5'-bromo-2'-deoxyuridine (BrdUrd), cytocentrifuged, fixed in ethanol, and then exposed to UV light to induce DNA strand breaks at the sites of incorporated BrdUrd. The 3' OH termini of the photolytically generated DNA strand breaks were labelled with BrdUTP in the reaction catalysed by exogenous terminal deoxynucleotidyl transferase (TdT), followed by FITC-labelled BrdUrd antibodies. DNA was counterstained with propidium iodide (PI). Due to differences in chromatin structure between the interphase and mitotic cells, the LSC identified the latter by virtue of their higher red (PI) fluorescence intensity values among all pixels over the measured cell. To confirm that the cells selected were indeed cells in mitosis, predominantly in metaphase, the recorded X-Y coordinates of selected cells were used to re-position the cell for their visual examination. From the time lapse analysis of percentage BrdUrd-labelled cells progressing through mitosis it was possible to calculate the duration of individual phases of the cell cycle. The duration of S (Ts) and G2 + M (TG2 + M) was 8 and 3 h, respectively, and the minimal duration of G2 (TG2) was 2 h. The cell cycle time (Tc) estimated for the cohort of the most rapidly progressing cells was 13 h. The ability to automatically and rapidly discriminate mitotic cells combined with the possibility of their subsequent identification by image analysis makes LSC the instrument of choice for the FLM analysis.

Bromodeoxyuridine↗
PubMed1996

A new flow cytometric approach to the analysis of DNA and a second parameter.

OBJECTIVE: To describe a new method of analyzing flow cytometry data by continuously variable gating (CVG), which is particularly useful for analysis of cellular DNA plus a second parameter, such as the proliferation marker Ki-67. STUDY DESIGN: CVG was compared with histogram subtraction (HS) using Overton's cumulative subtraction and with boxed gating (BG). Background suppression (thresholding) was used to remove outliers. The comparisons were carried out on a human bladder cancer cell line (T24) in various phases of cell proliferation and on disaggregated cells of fresh specimens of human prostatic and bladder carcinoma. With similar counts in control and test samples, data did not have to be normalized. RESULTS: In most cases histogram analysis by CVG was similar to that of BG and HS. However, when true positive values showed only a slight shift in fluorescence intensity, HS generated higher values than CVG. When true positive values exhibited strong fluorescence signals, HS could falsely include minor shifts in fluorescence values, and CVG became more accurate. CVG generated a continuous plot of percent positive events that could be superimposed on the DNA histogram, or a DNA histogram of positive events only could be created. This latter method demonstrated the DNA subpopulations containing most of the positive cells and improved discrimination of small subpopulations. CONCLUSION: The CVG method presents data in unique graphic form from flow cytometry assays of DNA and a second parameter. It is relatively objective and particularly useful in analyzing specimens with multiple DNA populations.

Cell Cycle↗
PubMed1995

Single-step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides: detection of apoptosis and bromodeoxyuridine incorporation.

The methods of in situ labeling of DNA strand breaks have been used to identify apoptotic cells and/or DNA replicating cells. While discrimination of apoptotic cells is based on the inherent presence of numerous DNA strand breaks in their chromatin, DNA proliferating cells can be discriminated by the selective DNA strand break induction by photolysis (SBIP) methodology at the sites that contain incorporated bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). In both instances, DNA strand breaks are labeled with biotin- or digoxygenin-conjugated deoxynucleotides, preferably in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase; fluorescein tagged avidin (streptavidin) or digoxygenin antibody is used in the second step of the reaction. In the present study, DNA strand break labeling was simplified by using directly labeled deoxynucleotides, in a single-step reaction. Cell fluorescence was measured by flow cytometry as well as by a microscope-based laser scanning multiparameter cytometer. Apoptotic cells in HL-60 cultures treated with camptothecin or in primary cultures of non-Hodgkin's lymphoma cells treated with prednisolone were easily identified utilizing BODIPY-conjugated dUTP (B-dUTP). Apoptotic cells were also recognized using fluorescein-conjugated dUTP or dATP, although the discrimination was more pronounced with B-dUTP. The single-step procedure, requiring fewer centrifugation steps, resulted in less cell loss compared to the two-step cell labeling technique.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis↗
PubMed1995

Comparison of fluorescence in situ hybridization and flow cytometric DNA ploidy analysis in paraffin-embedded prostatic adenocarcinoma specimens.

Cancer cell nuclei extracted from 15 paraffin-embedded radical prostatectomy specimens were examined with fluorescent in situ hybridization (FISH) and flow cytometry (FCM) for DNA ploidy. Repetitive chromosome-specific alpha-satellite DNA probes to centromeres 7 and 10 were used in the FISH assay. Results from FISH and FCM were compared and viewed in relation to clinical experience. Of the 15 tumors examined, 9 were hyperdiploid for at least one chromosome by FISH assay. Seven were aneuploid by FCM, showing a good correlation between the two methods (P = .006). Using either evidence of clinical progression or a postoperative prostate-specific antigen (PSA) level > 0.5 ng/mL by Hybritech assay to indicate the risk of clinical progression, 56% of FISH hyperdiploid tumors had a risk of progression of carcinoma versus 17% of FISH diploid tumors. A preoperative PSA level > 30 ng/mL had a marginal correlation with risk of progression (P = .05). We demonstrate that FISH is a useful tool for chromosomal analysis in paraffin-embedded tumor specimens.

Adenocarcinoma↗
PubMed1995

Flow cytometric characterization of proliferation-associated nuclear antigen (p105) during the cell cycle in normal lymphocytes and promyelocytic leukemia cells (HL-60).

Bivariate flow cytometric analysis of p105 expression and DNA content was performed in human lymphocytes and promyelocytic leukemia cells (HL-60). We also employed a new method of detecting DNA strand breaks associated with apoptosis by labeling the 3'-OH termini in the breaks with biotinylated dUTP in a reaction employing exogenous terminal deoxynucleotidyl transferase (TdT). Phytohemagglutin-stimulated proliferating lymphocytes express p105 maximally after 48 hours, similar to HL-60 cells in exponential growth phase. Antigen expression in G1 was notably heterogeneous in G1 phase of both cell types and highest in M-phase cells treated for 6 hours with vinblastine. However, the p105-DNA ratio changed very little. Cycloheximide did not affect P105 expression. Methotrexate decreased p105 expression. Camptothecin and teniposide induced apoptosis, but apoptotic cells still expressed p105. Dual-parameter measurement also demonstrated that TdT-positive apoptotic cells expressed p105 at a higher level than apoptotic TdT negative cells. The data on drug treatment suggest that expression of p105 may be useful in monitoring chemotherapeutic effects but not as a marker of cell death from apoptosis.

Camptothecin↗
PubMed1994

Display and correction of flow cytometry time-dependent fluorescence changes.

The resolution of measurements of cellular attributes by flow cytometry, based on differences in labeling by fluorescent markers, may be affected by variations in staining and sample stream flow that can occur during the course of data acquisition. We describe a method of display and correction of time-dependent changes by looking at the list mode data in chronological order.

Cell Separation↗
PubMed1994

Effects of suramin on expression of proliferation associated nuclear antigens in DU-145 prostate carcinoma cells.

Suramin is the first putative growth factor inhibitor in clinical trial that has demonstrated antitumor activity. Bivariate flow cytometric analysis of the effects of suramin on the cell cycle was performed on DU-145 prostate carcinoma cell line. The effects of suramin on the expression of proliferation associated antigens (p145, p120, PCNA, Ki-67, and cyclin A) were also studied. At concentrations of 50-500 micrograms/ml, suramin arrested cells in G1 and decreased S-phase moderately. The expression of p145, p120, PCNA, Ki-67 and cyclin A in these cells was reduced. The data suggest that suramin not only inhibits proliferation, but also reduced the expression of proliferation associated antigens. The expression of these antigens may be considered a means to monitor suramin treatment in vivo and in vitro.

Antigens, Neoplasm↗
PubMed1994

Detection of apoptosis-associated DNA strand breaks in fine-needle aspiration biopsies by in situ end labeling of fragmented DNA.

The predominant mode of either spontaneous or drug-induced death of cells in tumors is apoptosis. A flow cytometric method was developed in our laboratory to identify apoptotic cells, based on labeling DNA strand breaks, which appear as a result of extensive DNA cleavage by the apoptosis-associated endonuclease, with biotinylated dUTP in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The aim of this study was to reveal whether this methodology can be applied to human solid tumors sampled by fine-needle biopsy. Twenty-two tumors, consisting of 11 breast carcinomas; three metastatic anaplastic carcinomas; three adenocarcinomas of colon, endometrium, and lung; two metastatic lymph node squamous cell carcinomas of the larynx; and three malignant lymphomas were examined. It was possible to identify cells with DNA strand breaks in all these tumors. Extremely high variability in the proportion of cells with DNA strand breaks was observed between the individual tumors. In diploid tumors (n = 12) the percentage of cells with DNA strand breaks varied from 1% to 43%, and the mean value was 19%. In aneuploid tumors this percentage varied from 15% to 51% and the mean value was 37%. In the latter tumors the presence of cells with DNA strand breaks was limited to the DNA aneuploid cell population; very few diploid, presumably tumor infiltrating or stromal cells, showed the presence of DNA strand breaks. No correlation was observed between the percent of cells in S phase and those with DNA strand breaks. The data indicate that apoptosis is more frequent in populations of tumor cells than among normal cells of the same organs.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗
PubMed1994

Dietary beta carotene and lung cancer risk in U.S. nonsmokers.

BACKGROUND: Approximately 15% of all lung cancer deaths in the United States (about 22,350 deaths annually) may not be directly attributable to active cigarette smoking. Consumption of beta carotene, which is derived almost exclusively from intake of fruits and vegetables, has been associated with a reduced risk of lung cancer in smokers. However, studies examining this association in nonsmokers, particularly nonsmoking men, are limited. PURPOSE: The purpose of this study was to examine whether dietary factors including beta carotene and retinol are associated with a reduced risk for lung cancer in nonsmoking men and women. METHODS: A population-based, matched case-control study of lung cancer in nonsmokers was conducted in New York State from 1982 to 1985. Dietary interviews were completed for 413 individually matched case-control pairs of subjects. To determine whether the relationship between dietary intake from specific food groups and lung cancer differed by type of interview, smoking history, sex, age, or histologic type, we examined data on the case-control pairs from each subgroup separately. The intake of beta carotene and retinol was calculated as the weighted sum of the monthly frequencies of consumption of food items containing these nutrients, where the weights correspond to the nutrient content of a typical portion of the food items. RESULTS: Consumption of greens (P for trend < .01), fresh fruits (P for trend < .01), and cheese (P for trend < .05) was associated with a significant dose-dependent reduction in risk for lung cancer, whereas consumption of whole milk (P for trend < .01) was associated with a significant dose-dependent increase in risk. Use of vitamin E supplements was also protective (odds ratio = 0.55; 95% confidence interval [CI] = 0.35-0.85). Increased consumption of the following food groups was associated with a reduction in risk among females: vegetables (P for trend < .025), raw fruits and vegetables (P for trend < .005), and dairy products (P for trend < .025). In males, increased consumption of raw fruits and vegetables was associated with a reduced risk for lung cancer (P for trend < .005). Dietary beta carotene (OR = 0.70; 95% CI = 0.50-0.99), but not retinol (OR = 0.98; 95% CI = 0.82-1.17), was significantly associated with risk reduction. CONCLUSIONS: This is the largest study to date of dietary factors and lung cancer in nonsmokers; results suggest that dietary beta carotene, raw fruits and vegetables, and vitamin E supplements reduce the risk of lung cancer in nonsmoking men and women.

Adult↗
PubMed1994

Detection of occult micrometastases in the bone marrow of patients with prostate carcinoma.

A panel of three monoclonal antibodies that recognize membrane and cytoskeletal antigens expressed by epithelial cells (T16, C26, and AE-1) was used in a sensitive immunohistochemical assay to detect tumor cells in bone marrow aspirates from 20 patients with prostate cancer. Bone marrow aspirates from 2/9 (22%) patients with localized prostate cancer (stage B, 0/5; Stage C, 2/4), and 4/11 (36%) patients with metastatic prostate cancer (Stage D1, 0/7 patients; Stage D2, 4/4 patients) had antigen-positive cells in their bone marrow. The patients with localized disease had conventional examinations for metastases, including radioisotope bone scans and examination of bone marrow cytology, which were negative. The serum prostatic specific antigen (PSA) level appeared to correlate with the presence of micrometastases. Those patients with localized disease and antigen-positive cells in the bone marrow had an average serum PSA level of 26.6 ng/ml, while the average serum PSA level in patients without antigen-positive cells was 12.3 ng/ml. In addition, the number of antigen-positive cells detected appeared to correlate with the stage of disease; patients with Stage C prostate cancer had an average of 10 antigen-positive cells per one million bone marrow elements, while patients with Stage D2 disease had an average of 25 antigen-positive cells per one million bone marrow elements. We have demonstrated that immunohistochemical staining of bone marrow aspirates can detect occult bone marrow metastases in patients with apparently localized prostate cancer. Further follow-up of these and a larger number of patients will be require to determine the potential clinical significance of this finding.

Acid Phosphatase↗
PubMed1994

Effects of selected chemotherapeutic agents on PCNA expression in prostate carcinoma cell lines.

Bivariate flow cytometric analysis of proliferating cell nuclear antigen (PCNA) was performed on prostate carcinoma cell lines (PC-3, DU-145). For both cell lines 100% methanol fixation provided optimal fluorescence intensity of PCNA. The ratio of PCNA/DNA increased in late G1 through early S/phase, followed by a decrease in mid- and late S and enhancement in G2/M phase. PCNA expression was increased in G2/M phase cells treated for 48 h with vinblastine. A slight decrease in PCNA expression was observed with cyclohexamide treatment. Hydroxyurea induced an increase in S-phase fraction along with enhanced PCNA expression. Methotrexate and Adriamycin had little effect on the cell cycle compartments of PC-3 or DU-145; however, methotrexate decreased PCNA expression, while Adriamycin enhanced it. Cisplatin increased S-phase in both cell lines, increasing PCNA expression in PC-3 and decreasing it in DU-145 cells. The data on the effects of drug treatment point to a dissociation between PCNA expression and S-phase fraction as calculated from the DNA distribution. In some cases, e.g., the cisplatin studies, different effects were obtained in the two different cell lines treated with the same drugs. Whether changes in PCNA expression will provide more useful information than S-phase fraction for evaluation of potential antitumor drugs is not known.

Antineoplastic Agents↗
PubMed1994

The cell cycle effects and induction of apoptosis by 5-bromouridine in cultures of human leukaemic MOLT-4 and HL-60 cell lines and mitogen-stimulated normal lymphocytes.

5-Bromouridine (BrUrd) and several analogues of this antimetabolite exhibit antiviral activity and are potent inhibitors of the human immunodeficiency virus. The antitumour activity of BrUrd, however, in comparison with 5-fluorouridine or 5-fluorouracil, is less pronounced. Because BrUrd is incorporated into RNA and can be detected immunocytochemically and analysed by cytometry, it may prove useful as an RNA precursor to assay cell transcriptional activity. The aim of the present study was to evaluate the cell cycle effects of BrUrd incorporation in human lymphocytic leukaemic MOLT-4 and HL-60 cells and mitogenically stimulated normal peripheral blood lymphocytes. BrUrd suppressed cell proliferation and induced cell death; when measured after 72 h of treatment, the LD50 was 10 and 20 microM for HL-60 and MOLT-4, respectively, and LD90 was 100 microM, for both cell lines. BrUrd was maximally incorporated by the cells progressing through S phase of the cycle and the S phase cells were most severely perturbed by the drug, which was detected in RNA but not in DNA. Suppression of the S phase traverse of MOLT-4, HL-60 and normal lymphocytes was seen at > or = 30 microM BrUrd concentration. Also sensitive was the cell traverse through G2+M which, in the case of lymphocytes, HL-60 and MOLT-4 cells, was perturbed at > or = 30, > or = 200 and 500 microM BrUrd concentrations, respectively. Transition of lymphocytes from G0 to G1 was little affected at < 100 microM, and, although suppressed, was still observed even at 500 microM BrUrd concentration. Apoptosis of HL-60 cells and lymphocytes was observed at > or = 50 microM BrUrd after 24 h of incubation; death of MOLT-4 cells had mixed features of apoptosis and necrosis and resembled the 'mitotic' or 'reproductive cell death' as described in other cell systems. The cytostatic and cytotoxic effects of BrUrd should be taken into consideration when using this antimetabolite as an antiviral agent in the clinic or as an RNA precursor in assays of cell transcriptional activity.

Antimetabolites↗