Biomedical subjects

M R Melamed

Publications and source records attributed to M R Melamed.

At least 55 records · Page 3Linked to original sources

PubMed1994

Bivariate flow cytometric analysis of p53 and DNA content in hepatocellular carcinoma.

Bivariate flow cytometric analysis of p53 protein and DNA content was studied in archival specimens of hepatocellular carcinoma (HCC) from Chinese patients and corresponding benign liver tissues from a series of 51 patients at Sun Yat-sen University of Medical Sciences. Extracted nuclei were stained with the fluoresceinated monoclonal antibody PAb 1801, which recognizes human p53 protein (mutant and wild types). The nuclei were counterstained with the DNA stain propidium iodide. They were measured on an Ortho FC-200 flow cytometer and the data acquired and analyzed with an IBM 386 personal computer using Kusuda's Get Simple and List Simple software. Of the 51 hepatomas studied, 26 (51%) were p53 positive as compared with 4 (16%) of 24 samples of benign liver tissue from the same patients (P < .0257). The S-phase fraction of p53-positive HCC (12.3 +/- 8.8%) (SD) was significantly greater (P < .05) than for p53-negative HCC (7.4 +/- 7.2%). p53 Expression did not correlate with age, sex, alpha-fetoprotein, hepatitis B surface antigen, tumor size, tumor grade or survival rate. List Simple software permitted analysis of each specimen together with its isotype control (IgG1) on the same cytogram so that p53 expression could be determined separately for the diploid and aneuploid populations of aneuploid tumors and for tumor cells of diploid tumors in the various phases of the cell cycle. Since p53 (PAb 1801) expression can withstand formalin fixation and pepsin treatment of paraffin-embedded tissues, flow cytometric analysis of archival specimens is feasible, and clinical correlations such as these may be carried out in retrospective studies of other tumors.

Adult↗

PubMed1994

Current status of screening for lung cancer.

Three parallel studies on screening for lung cancer, supported by the National Cancer Institute, were carried out by the Mayo Clinic, Johns Hopkins Medical Institutions, and Memorial Sloan-Kettering Center from 1971 to 1982. No significant mortality improvement was established that could be attributed to cytologic screening examinations. Many stage I lung cancers were detected by radiographic screening, with excellent changes for long-term survival. Patients with stage I cancers who were operated on had significantly better survival rates than those who failed to undergo surgery. Statistical modeling, however, indicates that long-term annual screening of a high-risk population would decrease lung cancer mortality by no more than 18%.

Adenocarcinoma↗

PubMed1993

Screening for lung cancer. The Mayo Lung Project revisited.

BACKGROUND: The Mayo Lung Project (MLP) reported lung cancer incidence and mortality in a population offered chest radiographs and sputum cytologic screening examinations every 4 months and a population offered only the Mayo Clinic advice to undergo annual examination. No mortality benefit attributable to screening was observed after 6 years of observation and at least 1 year of follow-up. METHODS: The authors describe a simulation study designed to estimate from Mayo data the parameters in a mathematical model of the natural history of lung cancer and to estimate the potential benefit associated with periodic screening of high-risk people starting at 45 years of age. RESULTS: It was found that the mean duration of Stage I non-small cell lung cancer is at least 4 years and that rates of Stage I detectability and curability are less than 25% and 35%, respectively. CONCLUSIONS: A trial of the magnitude, duration, and contamination of the MLP would have a less than 20% probability of showing significant benefit from screening; however, long-term annual screening might result in a modest decrease in lung cancer mortality, ranging from 0% to 13%. A greater benefit would accrue from improved detection and treatment.

Aged↗

PubMed1993

Early lung cancer as a potential target for chemoprevention.

Carcinoma of the lung is the most common cause of death from cancer in the United States. In considering lung cancer for possible chemoprevention trials, we have analyzed the data collected by the collaborative NCI program on early lung cancer. The data indicate that at least 12 years of study of 80,000 people at risk for lung cancer (adult male cigarette smokers) would be required to establish a 25% reduction in squamous carcinoma of the lung. No intermediate markers of developing lung cancer are presently available to shorten the observation period. It is concluded that a study of the magnitude required is not feasible at the present time.

Adult↗

PubMed1993

p145 expression during the cell cycle in HL-60 cell line and normal human lymphocytes: effects of camptothecin, vinblastine, cycloheximide, actinomycin D, retinoic acid and DMSO.

Bivariate flow cytometric analysis of nucleolar antigen p145 was performed on quiescent and phytohemagglutinin-stimulated human lymphocytes and on a promyelocytic cell line (HL-60). Data were acquired on a FACScan flow cytometer and analyzed using LYSYS II. Quiescent lymphocytes did not express p145. PHA-stimulated proliferating lymphocytes expressed p145 maximally after 48 h, similarly to HL-60 cells in exponential growth. Antigen expression in G1 was notably heterogeneous in both cell types. The ratio of p145/DNA was highest in early S and decreased during mid and late S and remained low in G2M. p145 expression was lowest in M-phase cells treated for 6 h with vinblastine. Cycloheximide and actinomycin D had similar effects on p145 in HL-60 cells: expression of p145 gradually decreased from 1 to 6 h incubation in all phases of the cell cycle. Camptothecin did not decrease p145 expression and apoptotic cells from CAM-treated cultures still expressed p145. Retinoic acid and DMSO induced differentiation in HL-60 cells, and as this process progressed, p145 levels gradually fell until they approached isotype antibody control levels at 9 and 6 days, respectively. However, after 5 days treatment with 2 nM retinoic acid apoptotic cells appeared which still expressed p145. The data on drug treatment suggest that p145 exists in undifferentiated and proliferating cells and may not be a specific marker for malignancy, but may prove useful as a monitor of chemotherapeutic effects in cancer treatment.

Antigens, Nuclear↗

PubMed1993

Apoptosis of S-phase HL-60 cells induced by DNA topoisomerase inhibitors: detection of DNA strand breaks by flow cytometry using the in situ nick translation assay.

DNA topoisomerase I inhibitor camptothecin (CAM), topoisomerase II inhibitors teniposide (TN) and amsacrine (m-AMSA) induce apoptosis of HL-60 cells. One of the early events of apoptosis is DNA degradation, which occurs as a result of activation of the specific endonuclease. DNA strand breaks generated during this process were revealed, in the present study, by the in situ nick translation assay which was adapted to flow cytometry. In this assay, the incorporation of biotinylated dUTP by apoptotic cells was detected by the use of fluorescinated avidin, whereas simultaneous staining of DNA with propidium iodide made it possible to correlate the appearance of DNA strand breaks with cell position in the cell cycle. The breaks were detected as early as 90 min after the initial cell contact with CAM, and they were limited to cells in the S phase of the cell cycle. At that early stage of apoptosis DNA was not yet extractable from the cells; the loss of DNA from S-phase cells could not be seen, by flow cytometry, during the initial 2 h of incubation with CAM. DNA strand breaks induced by TN and m-AMSA also occurred preferentially in S-phase cells. The data indicate that DNA strand breaks resulting from activation of endonuclease in HL-60 cells treated with DNA topoisomerase I or II inhibitors can be conveniently measured using the in situ nick translation assay. This assay has certain advantages over other methods of identification of apoptotic cells by flow cytometry, such as providing direct evidence of DNA damage and offering the opportunity to correlate DNA damage with cell position in the cell cycle. The method may be of interest in clinical oncology where testing tumor response (by DNA degradation) to DNA topoisomerase inhibitors or other treatments may be of prognostic value.

Amsacrine↗

PubMed1993

Proliferating cell nuclear antigen in archival surgical specimens of malignant lymphoma and metastatic carcinoma: immunohistochemical and flow cytometric analysis.

Immunohistochemical and flow cytometric multiparameter analysis of proliferating cell nuclear antigen (PCNA) was performed on fifteen formalin fixed, paraffin embedded lymph nodes with malignant lymphoma (eleven non-Hodgkin's lymphomas, four Hodgkin's lymphomas), and fifteen lymph nodes with metastatic carcinomas. A general concordance between PCNA measurement by both methods has been observed: the percentage of positively stained cells in tissue sections correlated well with the percentage of cells expressing this antigen in cell suspensions (r = 0.76). Both diploid and aneuploid tumors expressed PCNA, and a correlation between PCNA and the percent cells in S-phase was evident in both: in PCNA-positive tumors the mean percent of cells in S-phase was 16.5%, and in PCNA-negative tumors, 5.9%. The data indicate that PCNA can be detected in formalin-fixed tissues by either classic immunohistochemical analysis or by flow cytometry.

Adolescent↗

PubMed1993

[Programmed death of cells (apoptosis)].

Apoptosis is a mode of cell death defined by characteristic morphological, biochemical and molecular changes. It was first described as a "shrinkage necrosis", and then this term was replaced by apoptosis to emphasize its role opposite mitosis in tissue kinetics. During apoptosis the cell decrease in size, loose contact with neighboring cells, and loose specialized surface elements such as microvilli and cell-cell junctions. A shift of fluid out of the cells causes cytoplasm condensation, which is followed by convolution of the nuclear and cellular outlines. In later stages of apoptosis the entire cell becomes fragmented, forming a number of plasma membrane-bounded apoptotic bodies which contain nuclear and or cytoplasmic elements. The ultrastructural appearance of necrosis is quite different, the main features being mitochondrial swelling, plasma membrane breakdown and cellular disintegration. Apoptosis occurs in many physiological and pathological processes. It plays an important role during embryonal development as programmed cell death and accompanies a variety of normal involutional processes in which it serves as a mechanism to remove "unwanted" cells. Apoptosis is associated with prostate atrophy after castration or atrophy of the adrenal cortex and thymus after administration of glucocorticoids. Apoptosis is involved in elimination of CD4 T lymphocytes in the course of HIV infection. The interest in apoptosis in oncology stems from the fact that it occurs in tumors, spontaneously as well as triggered by different antitumor drugs, radiation or after withdraw of growth factors. Spontaneous apoptosis may play a role in evolution of tumor malignancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis↗

PubMed1993

Pathology and staging of urothelial tumors of the kidney and ureter.

Urothelial tumors of renal pelvis and ureter are rare but clinically important neoplasms. They are histologically and cytologically similar to tumors of the bladder. As in the bladder, tumor stage and grade are the most important prognostic parameters. The TNM and AJCC staging systems are described, as are criteria and other lesions that may be mistaken for urothelial tumors.

Aged↗

PubMed1993

Induction of DNA strand breaks associated with apoptosis during treatment of leukemias.

A new flow cytometric method is described to detect 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. The method has been applied in studies on leukemic HL-60 and MOLT-4 cell lines to reveal whether it is specific to apoptotic cells, and whether it can be used in the clinic to detect DNA breakage in leukemic cells during chemotherapy. There was labeling of mononuclear cells in peripheral blood of all 11 patients studied during chemotherapy for acute lymphoblastic, acute myelogenous, or chronic myelogenous leukemia (ALL, AML, or CML) in blastic crisis, indicating induced DNA damage; the number of labeled cells increased from 1-8% before treatment up to 80% during the course of treatment. The DNA topoisomerase inhibitors mitoxantrone, VP-16 (etoposide), and m-AMSA (amsacrine) were more effective in inducing DNA breaks than was hydroxyurea or cytosine arabinoside (AraC). Cells with DNA breaks were identified in peripheral blood for up to 5 days following administration of Mitoxantrone and VP-16. In the case of DNA aneuploid leukemias, the DNA breaks were predominant in the aneuploid cell subpopulations, whereas presumably non-neoplastic diploid cells were unlabeled. In one case of ALL there were two distinct subpopulations of aneuploid cells: one responded to the treatment (by DNA breakage) and the other was non-responding. Thus, cells undergoing apoptosis can be detected by this method of labeling DNA strand breaks and the technique is applicable for analysis of response of leukemic cells to chemotherapy. With this method it may be possible to identify tumor cell sensitivity or resistance to particular drugs early in the course of treatment.

Adult↗

PubMed1992

Cell cycle-related expression of p120 nucleolar antigen in normal human lymphocytes and in cells of HL-60 and MOLT-4 leukemic lines: effects of methotrexate, camptothecin, and teniposide.

Expression of the proliferation-associated nucleolar antigen p120 was studied by flow cytometry in human quiescent and phytohemagglutinin-stimulated lymphocytes, as well as in human lymphocytic (MOLT-4) and promyelocytic (HL-60) cell lines. Bivariate analysis of p120 and DNA content made it possible to correlate p120 expression with cell position in the cycle. Proliferating lymphocytes and MOLT-4 and HL-60 cells had a similar pattern of p120 expression. Populations of G1 cells, in all three cell types, were very heterogenous with respect to p120, and a threshold in G1 was observed. The cells with a p120 level below the threshold value did not enter S phase. An increase in p120 was observed during progression through S phase, and the antigen was maximally expressed in G2 cells. The p120/DNA content ratio, however, was highest in late G1 cells (G1B) and was declining during S and G2. The data thus suggest that p120 may be degraded during mitosis and that the postmitotic cells inherit little, if any, of this protein; the antigen then accumulates predominantly during G1, and must reach a threshold level to enable the cells to enter S phase. Antigen p120 could not be detected in noncycling lymphocytes nor in HL-60 cells induced to myeloid differentiation by growth in the presence of dimethyl sulfoxide. Treatment of MOLT-4 cells with pharmacological concentrations of methotrexate, camptothecin, or teniposide induced cell arrest in S or G2; expression of p120 in the arrested cells was unchanged from that of untreated MOLT-4 controls at the same phase of the cycle. The level of p120 was minimal in MOLT-4 or HL-60 cells arrested in M phase by vinblastine, but vinblastine had no effect on p120 fluorescence of interphase cells. Camptothecin or teniposide induced apoptosis selectively in S phase of HL-60 cells; apoptotic cells from camptothecin-treated cultures, however, despite the marked nucleolysis, still expressed p120. The data on the drug-treated cells indicate that the p120 level in tumors of patients may be used as a marker of tumor/malignancy even in clinical samples obtained during treatment.

Antigens, Neoplasm↗

PubMed1992

Increase in acridine orange (AO) fluorescence intensity of monocytes cultured in plastic tissue culture plates as measured by flow cytometry.

Although the green-red fluorescence of AO is an accepted measure of DNA-RNA content, respectively, it is actually a measure of the fluorescence of dye bound to nucleic acids, and may vary with changes in accessibility to the dye. It has been shown for example that extraction of nuclear proteins results in a marked increase in DNA stainability. Moreover, in certain cell systems the binding of fluorochromes correlates with structural modifications in chromatin that accompany cell differentiation. We report here that changes in green & red fluorescence intensity also occur in long-term monocyte cultures. The increased red fluorescence intensity observed in cultured monocytes may reflect ribosomal RNA synthesis and the increased green fluorescence enhanced AO accessibility to DNA due to changes in chromatin organization. We compared cultured monocytes from bladder cancer patients and healthy donors. The results indicate a small but statistically significantly greater increase in mean green & red fluorescence of cultured monocytes from the cancer patients. These fluorescence variations may indicate differences in the immunologic status of cancer patients and/or be related to disease state.

Acridine Orange↗

PubMed1992

DNA stainability in aneuploid breast tumors: comparison of four DNA fluorochromes differing in binding properties.

The aim of this study was to evaluate whether or not the differences in chromatin structure between diploid stromal cells or lymphocytes, which are often used as DNA ploidy standard, and aneuploid breast tumor cells can significantly affect the estimates of the DNA index of these tumors. To this end, the DNA content estimates of 34 aneuploid breast tumors, differing in size, degree of differentiation, and presence or absence of estrogen and progesterone receptors and metastases, were compared using four common DNA fluorochromes: DAPI, Hoechst 33342, propidium iodide, and acridine orange. These dyes differ in their mode of interaction with DNA (binding to minor groove or intercalation) and for each of them binding to DNA is restricted to a different degree by nuclear proteins. It was expected, therefore, that if differences in chromatin structure play a role in DNA content estimates, the DNA index of the measured tumors may vary depending on the dye. The cell nuclei were isolated from the tumors using a detergent-based procedure and stained with each of the dyes and the DNA index was estimated using peripheral blood lymphocytes as a DNA content standard. For each of the tumors, the DNA index estimates with all four dyes correlated very well. When the results obtained with individual dyes were compared in pairs, the correlation coefficients (r) of DNA indices were all above 0.96 (correlation at p less than 0.001). The best concordance was seen between specimens stained with Hoechst 33342 and DAPI (r = 0.99), and the least between those stained with Hoechst 33342 and propidium iodide (r = 0.96). The data indicate that DNA content analysis of unfixed nuclei, utilizing the above fluorochromes, is not significantly biased by differences in chromatin structure of the measured cells.

Acridine Orange↗

PubMed1992

Papillary tumors of the bladder.

The most common neoplasms of the urinary bladder are papillary tumors that vary histologically and cytologically from very well differentiated to highly anaplastic patterns. Biologic behavior of these tumors is closely correlated with morphology, so that cytologically benign tumors (papillomas) are benign in behavior, and increasing anaplasia is associated with increasing clinical aggressiveness. By this definition, 20% or more of bladder tumors should be classified as papillomas. The development of carcinoma occurs in a series of steps, progressing through atypia and carcinoma in situ to invasion. Finally, evidence is presented to show that invasive carcinoma often begins from areas of flat carcinoma in situ associated with, but not within, co-existing papillary tumors.

Carcinoma, Papillary↗

PubMed1992

Cytology.

Exfoliative cytology is a relatively sensitive, highly specific technique for the detection and diagnosis of bladder cancer, and for monitoring conservative treatment. Cytologic diagnosis is based on cellular changes in DNA content, chromatin structure, and metabolic activities which are characteristic of cancer cells and can be recognized in desquamated isolated cells or cell clusters. The cytologic techniques are most effective in identifying low stage flat or papillary carcinoma, but not cytologically benign papillomas or ulcerating invasive cancers that do not easily shed cancer cells. With the growing number of pathologists who, in recent years, have become skilled cytopathologists, urinary tract cytology has developed into an essential diagnostic tool for the urologist.

Cytodiagnosis↗

PubMed1992

The effect of surgical treatment on survival from early lung cancer. Implications for screening.

We assessed the effect of surgery on survival from stage I non-small-cell lung cancer based on data collected in these screening programs. The majority of patients diagnosed in each program were treated by surgical resection, but 5 percent of the Sloan-Kettering group, 21 percent of the Hopkins group and 11 percent of the Mayo group failed to receive surgical treatment. Approximately 70 percent of the stage I patients in each program who were treated surgically survived more than five years, but there were only two five-year survivors among those who did not have surgery. We conclude that patients with lung cancers detected in stage I by chest x-ray film and treated surgically have a good chance of remaining free of disease for many years. Those stage I lung cancers which are not resected progress and lead to death within five years. Therefore, every effort should be made to detect and treat lung cancer early in high-risk populations.

Age Factors↗

PubMed1992

Immunopathologic analysis of human urinary bladder cancer. Characterization of two new antigens associated with low-grade superficial bladder tumors.

The authors have further characterized the normal human tissue distribution and tumor expression of two highly restricted tumor-associated antigens, detected by mouse monoclonal antibodies M344 and 19A211, which are primarily expressed on low-grade superficial urinary bladder tumors. This study was conducted using immunohistochemical staining of frozen and deparaffinized sections of human normal and tumor tissues. The antigens are stable and well preserved on deparaffinized tissue sections. M344 antibody identifies a high-molecular-weight determinant on a cytosolic protein component of over 300,000 Mr. This antigen was not detected on any normal tissue analyzed, including 14 specimens of normal urothelium and 22 cases of cystitis; however, M344 was positive in 74.5% of Ta-T1 tumors and 11% of T3-T4 tumors. 19A211 antibody identifies a sialylated epitope on a cytoplasmic protein complex of 100,000 to 200,000 Mr. This antigen also was expressed preferentially on low-grade superficial bladder tumors (77% Ta-T1) and less frequently on deeply infiltrating tumors (10% T3-4). 19A211 was negative on all normal cells tested, with the exception of umbrella cells, in approximately 25% of the normal urothelium and cystitis specimens studied. Either one or both of these tumor-associated antigens are detected in approximately 80% of low-grade papillary superficial tumors and carcinoma in situ of the urinary bladder. The expression of these antigens on a subset of low-grade bladder tumors, known to progress in only about 10% of cases, suggests that phenotypic differences may reflect biologic potential. Beyond their possible biologic significance, antibodies M344 and 19A211 may provide clinically useful probes for early detection and stratification of urinary bladder tumors.

Antibodies, Monoclonal↗

PubMed1991

DNA/RNA content and proliferative fractions of colorectal carcinomas: a five-year prospective study relating flow cytometry to survival.

In our prospective study, flow cytometric analysis of cellular DNA and RNA content was performed on unfixed fresh specimens of colorectal adenocarcinoma taken from 176 patients. Of the 176 tumors, 113 (64%) were aneuploid. There was no correlation between aneuploidy and tumor stage, grade, location, or size. After a median follow-up of 5.6 years, no correlation between DNA or RNA content and patient survival was found. DNA content alone was not an independent prognostic factor when the colorectal carcinomas were segregated by curable and incurable stages. However, normal mucosa, diploid tumors, and aneuploid tumors showed progressively higher proliferation and higher RNA and DNA indices. Proliferative fraction--defined as the percentage of cells in S + G2 and M phases of the cell cycle--was significantly related to ploidy and to Dukes' stage. Despite these correlations, we did not detect a significant influence of proliferative fraction on survival when patients were segregated above or below the mean proliferative fraction for all tumors. More accurate methods of identifying the proliferative fraction of tumor cells are currently being pursued. While the role of flow cytometry in the evaluation and management of patients with colorectal carcinoma is still undefined for a number of other cellular parameters, it seems unlikely that DNA index, RNA index, or the proliferative fractions calculated from the DNA histogram, will, of themselves, represent independent prognostic factors.

Adenocarcinoma↗