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Biomedical subjects

M R Melamed

Publications and source records attributed to M R Melamed.

At least 19 recordsLinked to original sources
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↗
PubMed1991

Blood group and blood-group-related antigens in normal pancreas and pancreas cancer: enhanced expression of precursor type 1, Tn and sialyl-Tn in pancreas cancer.

Expression of blood-group antigens A, B, Le(a), sialyl-Le(a) (sLe(a)), Le(b), Le(x), Le(y), precursor type I, Tn and sialyl-Tn (sTn) was examined in non-neoplastic pancreas (n = 37) and pancreas cancer (n = 21) using mouse monoclonal antibodies (MAbs). Immunohistochemical assays were performed on sections of paraffin-embedded tissues using the avidin-biotin complex method. In normal pancreas, antibodies detecting Le(a), sLe(a) and Tn reacted with ductal epithelium, and antibodies detecting A and B reacted with acini and ducts, independently of secretor status. Le(x) was weakly expressed in ducts and acini, and sTn could not be detected in normal pancreas. Expression of Le(b), Le(y) and precursor type I was regulated by secretor status: Le(b) and Le(y) were expressed in ducts of secretor and Le(a-b-) individuals, but not in ducts of non-secretors; precursor type I was weakly expressed in acini and ducts of non-secretors and Le(a-b-) individuals, and was absent in acini and ducts of secretors. The following alterations in the expression of blood-group antigens were observed in pancreas cancer: (1) enhanced expression of Le(x), Tn and sTn; (2) enhanced expression of precursor type I independently of secretor status; (3) loss of regulation of Le(b) by the secretor gene; (4) decreased expression of Le(y). The weak expression of precursor type I. Tn and sTn in non-neoplastic pancreas, and their stronger expression in pancreas cancer, suggests that up-regulation of their expression is associated with malignant transformation of pancreatic duct cells.

ABO Blood-Group System↗
PubMed1991

Precision of DNA flow cytometry in inter-institutional analyses.

A Bladder Cancer Flow Cytometry Network study has been carried out to further identify and quantify sources of inter- and intra-laboratory variability. Replicate samples containing four mixtures of peripheral blood lymphocytes and aneuploid cell lines were distributed together with reference standards to six laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Two of each of the four sample types and a reference standard were analyzed by each laboratory on 3 separate days to obtain cellular DNA distributions. DNA index (DI) and hyperdiploid fraction (HDF) were calculated for each histogram using an automated technique. The results showed significant inter- and intra-laboratory differences. Results were evaluated by a two-way analysis of variance to estimate components of the overall variation attributable to individual sources. Error variation was found to be the major component of random variation. Specimen means were also compared for each laboratory. No significant differences were noted in mean DI for similar specimens; however, agreement in HDF between similar specimens was lacking in most laboratories. Prediction intervals were computed to estimate the range of values expected for a single specimen based on the analysis of the previous six. Prediction intervals for DI were quite good while those for HDF were troublesome due to wide variation. The results of these studies indicate that intra- and inter-laboratory variability are high enough that results for a single sample may not be sufficiently precise to allow comparison to results obtained in other laboratories.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1991

Simultaneous detection of fluorescent in situ hybridization and in vivo incorporated BrdU in a human bladder tumour.

We have used fluorescent in situ hybridization and simultaneous in vivo bromodeoxyuridine labelling of a solid bladder cancer to examine tumour cell subsets for possible proliferative growth differences. In this dual-labelled preparation, most tumour cell nuclei exhibited monosomy 9, consistent with reported karyotypes of bladder cancer. Incorporated bromodeoxyuridine was visualized with a fluoresceinated antibody in 5-6 per cent of the tumour cells, concordant with S-phase estimates by cell cycle analysis of the flow cytometric DNA histogram. A majority of the bromodeoxyuridine-positive cells also carried the monosomy 9 chromosome abnormality. This is the first report to demonstrate the feasibility of combined in situ hybridization and detection of bromodeoxyuridine incorporated in vivo in human tumour cells in order to provide information on the growth rate of specific subsets of tumour cells identified by chromosomal constitution.

Bromodeoxyuridine↗
PubMed1991

Retention of the mitochondrial probe rhodamine 123 in normal lymphocytes and leukemic cells in relation to the cell cycle.

The cationic fluorochrome rhodamine 123 (R123) is specifically taken up by mitochondria of live cells where it is retained due to the mitochondrial transmembrane potential. After pulse exposure of human normal quiescent or proliferating lymphocytes, human lymphocytic leukemic MOLT cells, and mice leukemic L1210 cells to 10 micrograms/ml of R123, the dye release was studied using flow cytometry. Two distinct phases of R123 release, each following first-order kinetics, were apparent; the half-time of retention for the rapidly and slowly released fractions of R123 was 0.8-1.1 and 2.8-4.2 h, respectively. Simultaneous supravital cell staining with R123 and Hoechst 33342 made it possible to correlate retention of R123 with cell position in the cell cycle. No significant differences were observed in the rate of R123 release from cells in G1 vs S or vs G2 + M phases of the cycle. The data rule out a possibility that the release of R123 is due to periodic depolarization of the mitochondria in the cell as may be postulated by cell cycle models that assume a transient passage of cells through resting phase following division. The observed similar rates of R123 release regardless of cell type or cell cycle phase suggest that the factors affecting the exchange are similar in normal lymphocytes vs leukemic cells and unrelated to cell proliferation rate or phase of the cell cycle. Two distinct rates of R123 release indicate the presence of two kinds of binding sites differing in affinity to the dye.

Cell Cycle↗
PubMed1991

Immunohistochemical analysis of neural cell adhesion molecules. Differential expression in small round cell tumors of childhood and adolescence.

The neural cell adhesion molecule (NCAM) was discovered in a search for cell surface antigens of chicken neurons that contribute to cell adhesion and pattern formation during development. Homologous adhesion molecules have been identified in several species, including humans. In this immunohistochemical study, the authors examine the role of human NCAM in tumor diagnosis. The authors used a monoclonal antibody (MAb), 5.1H11, to examine NCAM immunoreactivity in frozen sections of more than 450 tumors, including more than 80 small round cell tumors (SRCT) of childhood and adolescence (neuroblastomas, Ewing's sarcomas [ES], peripheral neuroepitheliomas [PN], primitive neuroectodermal tumors [PNET], esthesioneuroblastomas, malignant ectomesenchymoma, medulloblastomas, small cell osteosarcomas, mesenchymal chondrosarcomas, embryonal rhabdomyosarcomas, and lymphomas). The authors show that 1) neuroblastomas and primary brain tumors are NCAM+; 2) ES, most PN/PNETs, and melanomas are NCAM-; 3) embryonal rhabdomyosarcomas and various other sarcomas are NCAM+; 4) neuroendocrine tumors are NCAM+; 5) subsets of carcinomas of kidney, ovary, lung and other organs are NCAM+; and 6) lymphoid tumors are NCAM-. Tests with normal fetal and adult tissues indicate that these findings reflect only in part the NCAM phenotypes of corresponding normal tissues. Notably the NCAM- phenotype of ES and PN/PNET is not explained by current histogenetic models for these tumors, which suggest a primitive neuroectodermal origin. Finally the authors show that NCAM expression among SRCT has an inverse relationship with the expression of p30/32MIC2, a cell surface antigen of ES and PN/PNET detected with MAb HBA71. These results suggest that immunohistochemical assays for NCAM and p30/32MIC2 expression may aid in the further characterization of SRCT of childhood and adolescence.

Antibodies, Monoclonal↗
PubMed1991

Effect of swainsonine on interleukin-2 alpha chain receptor expression and proliferation of human lymphocytes.

Swainsonine, an inhibitor of mannosidase II, involved in N-linked glycoprotein processing, modifies expression of cell surface receptors. This alkaloid has strong anti-metastatic and immunomodulatory activity; it enhances stimulation of lymphocytes triggered by concanavalin A (ConA) but suppresses stimulatory effects of phytohaemagglutinin (PHA). We presently observe that swainsonine decreases expression of the interleukin-2 (IL-2) receptor (IL-2R) on PHA-stimulated human peripheral blood lymphocytes, measured by binding of a monoclonal antibody that recognizes the 55-kD glycoprotein subunit (alpha) of this receptor. Proliferation of the PHA-stimulated lymphocytes is suppressed by swainsonine, which manifests in the decreased proportion of both cells entering G1 (from G0) and those progressing through S. G2 and M. This suppression can be overcome by addition of IL-2 into cultures. In contrast, swainsonine has no effect on IL-2R expression and stimulation (cell cycle progression) of lymphocytes triggered by the monoclonal antibody OKT3. The data suggest a possibility that the observed swainsonine effects on lymphocytes stimulated by PHA are mediated via surface receptors other than IL-2R. These receptors may appear prior to IL-2R and be also involved in cell stimulation by PHA but not by other mitogens.

Alkaloids↗
PubMed1990

DNA in situ sensitivity to denaturation in bladder cancer and its correlation with tumor stage.

DNA content and sensitivity of DNA in situ to denaturation by acid were analyzed by flow cytometry of cell nuclei freshly isolated from the bladder tumors of 32 patients and were compared with normal urothelium of 8 subjects. DNA sensitivity to denaturation was assessed in RNase treated cells by acridine orange metachromasia following partial denaturation with hydrochloric acid; the extent of denatured DNA is given as an index (alpha t), representing the ratio of single stranded to total DNA per nucleus. Of the low stage tumors (papillomas, Ta, Tis, T1) 11 of 18 (61%) were aneuploid. Of the high stage tumors (T2 and T3a) 11 of 14 (79%) were aneuploid. DNA in nuclei of normal transitional epithelium was very sensitive to denaturation, as was papilloma, characterized by nuclear alpha t indices of 0.73 +/- 0.01 (SD) and 0.73 +/- 0.04, respectively. Nuclear DNA of noninvasive carcinomas (Ta, Tis) was significantly more resistant to denaturation (alpha t = 0.69), and DNA of invasive carcinomas was most resistant, ranging from alpha t = 0.61 (T1 tumors) to alpha t = 0.59 (T2 tumors) to alpha t = 0.57 (T3 tumors). High stage tumors as a group (T2, T3) had significantly different (lower) alpha t values than low stage tumors (Ta, Tis, T1). In model cell culture systems it is known that a decrease in alpha t index, i.e., greater resistance to denaturability, occurs as cells transit from resting phase into the cell cycle. Whether the alpha t index can be used to estimate resting vesus cycling cells of human tumors is still speculative; changes in DNA denaturability also are known to occur with changes in chromatin structure during cell differentiation and in transformation. However, the empirical relationship between alpha t index and tumor stage, of itself, may prove clinically useful in identifying more advanced and perhaps more aggressive tumors.

Biopsy↗
PubMed1990

Lung cancer and exposure to tobacco smoke in the household.

BACKGROUND: The relation between passive smoking and lung cancer is of great public health importance. Some previous studies have suggested that exposure to environmental tobacco smoke in the household can cause lung cancer, but others have found no effect. Smoking by the spouse has been the most commonly used measure of this exposure. METHODS: In order to determine whether lung cancer is associated with exposure to tobacco smoke within the household, we conducted a population-based case--control study of 191 patients with histologically confirmed primary lung cancer who had never smoked and an equal number of persons without lung cancer who had never smoked. Lifetime residential histories including information on exposure to environmental tobacco smoke were compiled and analyzed. Exposure was measured in terms of "smoker-years," determined by multiplying the number of years in each residence by the number of smokers in the household. RESULTS: Household exposure to 25 or more smoker-years during childhood and adolescence doubled the risk of lung cancer (odds ratio, 2.07; 95 percent confidence interval, 1.16 to 3.68). Approximately 15 percent of the control subjects who had never smoked reported this level of exposure. Household exposure of less than 25 smoker-years during childhood and adolescence did not increase the risk of lung cancer. Exposure to a spouse's smoking, which constituted less than one third of total household exposure on average, was not associated with an increase in risk. CONCLUSIONS: The possibility of recall bias and other methodologic problems may influence the results of case-control studies of environmental tobacco smoke. Nonetheless, our findings regarding exposure during early life suggest that approximately 17 percent of lung cancers among nonsmokers can be attributed to high levels of exposure to cigarette smoke during childhood and adolescence.

Adolescent↗
PubMed1990

Targeted cytogenetic analysis of gastric tumors by in situ hybridization with a set of chromosome-specific DNA probes.

Fluorescent in situ hybridization (FISH) with biotinated chromosome-specific repetitive DNA probes was used for the cytogenetic study of ten gastric adenocarcinomas. All tumors (eight male, two female patients) were histologically moderately or poorly differentiated and nine of ten had metastasized to regional lymph nodes. The authors applied a set of satellite DNA probes, specific for chromosomes 1, 7, 17, X, and Y in order to detect numerical chromosome aberrations in freshly isolated tumor cell nuclei. Normal diploid human lymphocyte nuclei and, in a number of cases, normal gastric mucosa served as controls. Parallel with the hybridization experiments DNA flow cytometric study of acridine orange (AO)-stained tumor cells was carried out. By means of FISH the authors found seven cases to be aneuploid, the other three cases appeared diploid. This was confirmed by DNA flow cytometric analysis with AO. Furthermore, loss of the Y chromosome in a high percentage of cells was seen by FISH in six of eight tumors from male patients. In the other two male samples a possible loss was observed in a small proportion of cells (15%). In three patients from whom the authors had normal gastric mucosa the Y loss was restricted to the tumor cells. These data indicate that in situ hybridization with chromosome-specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of solid human tumors, at least with respect to the detection of numerical chromosome abnormalities.

Adenocarcinoma↗