Biomedical subjects

K D Bauer

Publications and source records attributed to K D Bauer.

At least 73 records · Page 4Linked to original sources

PubMed1987

Experimental parameters and a biological standard for acridine orange detection of drug-induced alterations in chromatin condensation.

We investigated a number of sample-preparative parameters for use of flow cytometry to detect chromatin condensation in cells stained with acridine orange after DNA in situ is partially denatured by acid treatment. Stability and data reproducibility for both control and drug-treated ME-180 and HT-29 cells were assessed over: a range of cell concentrations in 2.56 X 10(-5) M acridine orange; 15 days of storage in fixative; various times between RNase digestion and staining; and increasing times between staining and analysis. Listmode data for red and green fluorescence were collected and mean fluorescence intensities of G1, S, and G2 subpopulations of HT-29 and ME-180 cells were computed. These were normalized to data from HeLa-S3 cells and fluorescent microspheres to control for inter-experiment variations in staining and instrumental parameters, respectively. The normalized red and green fluorescence data were used to calculate alpha 1 for G1 cells [alpha t = red fluorescence/(total fluorescence)]. Exponentially growing HeLa-S3 cells were a very consistent and reproducible biological standard to control for fixation and staining variability. Mean fluorescence intensities of control and difluoromethylornithine-treated (i.e., polyamine depleted) cells remained stable and reproducible across all tested ranges for cell concentration, storage in fixative, and time after RNase digestion. This technique can thus be used to evaluate difluoromethylornithine-induced changes in chromatin condensation of samples stored for as long as 2 weeks and analyzed all on 1 day.

Acridine Orange↗

PubMed1987

Quantitative analysis of a nuclear antigen in interphase and mitotic cells.

The quantification of an interchromatin-associated antigen, designated p 105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p 105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p 105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p 105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p 105 immunofluorescence. The data reveal that the increased levels of anti-p 105 immunofluorescence in conjunction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p 105 throughout the cell cycle is discussed.

Antigen-Antibody Complex↗

PubMed1987

Modulation of the nuclear antigen p105 as a function of cell-cycle progression.

The characterization of the proliferation-associated nuclear antigen designated p105 in quiescent and proliferating lymphocytes is described. Through the use of novel flow cytometric and cell-sorting strategies the intracellular content of p105 was assessed in situ on a per cell basis. These analyses demonstrated the presence of multiple cellular subpopulations within the cell cycle differing significantly in p105 content. The data revealed that the flow cytometric quantitation of p105 levels may effectively discriminate cycling from noncycling cells. Immunogold electron microscopy revealed that the modulation of this interchromatin-associated antigen was correlated with a significant degree of nuclear restructuring. In conjunction with cell sorting, immunogold electron microscopy and immunoblot controls demonstrated that the cell-cycle-related modulation in p105 cannot be accounted for by increased cellular mass or antigen sequestration. The significance of these controls and of the potential role of p105 in cellular proliferation is discussed.

Autoantigens↗

PubMed1987

DNA aneuploidy in Hodgkin's disease. A multiparameter flow-cytometric analysis with cytologic correlation.

In 15 cases of Hodgkin's disease, the authors studied the DNA content of isolated nuclei from deparaffinized tissue by using multiparameter flow cytometry. An antinucleolar antibody preparation was employed as well as a secondary antibody that had been conjugated with fluorescein isothiocyanate. By simultaneously quantitating nucleolar fluorescence and DNA content, rare but distinct aneuploid populations were detected among the nuclei with brightly stained nucleoli. DNA aneuploidy was found in each case when this multiparameter analysis was used, but was detected in only 1 case when DNA content was analyzed alone. With the multiparameter analysis, two to four aneuploid populations were found in each case. These populations exhibited incremental duplications of DNA content that suggested endopolyploidy, ie, replication of DNA without accompanying nuclear division. The aneuploid stem line was hypodiploid or hypotetraploid in 6 cases, hyperdiploid in 7 cases, and near-triploid in 2 cases. These various abnormalities in ploidy showed only some correlation with histologic subtypes. Cell sorting showed that some nuclei with more than four or nearly eight times normal DNA content resembled nuclei of typical Reed-Sternberg cells. Many nuclei with an intermediate aneuploid DNA content resembled nuclei of mononuclear Reed-Sternberg cells. The near-diploid and near-triploid nuclei corresponded to nuclei of cells which were not readily recognizable as neoplastic in histologic sections. It is concluded that multiparameter analysis of DNA content can provide further insights into the neoplastic cells in Hodgkin's disease and may offer an objective basis for studying heterogeneity in this disorder.

Aneuploidy↗

PubMed1987

Recent progress in clinical quantitative cytology.

The last several years have seen changes in quantitative cytologic procedures that have significantly expanded the clinical research and diagnostic applications of quantitative cytologic analysis. Intact nuclei can be isolated from formalin-fixed paraffin-embedded tissue, and the DNA distribution of these nuclei can be analyzed either with flow cytometry or with slide-based image-processing techniques. In addition, more informative DNA analysis has been achieved using antibodies to bromodeoxyuridine-modified DNA, proliferation-associated nuclear antigens, or tissue-type-specific cytoplasmic antigens as second stains for two-parameter analysis. Improvements in both staining and instrumentation have also increased the usefulness of flow cytometry in analysis of "rare events," ie, cell types comprising less than 0.1% to 0.5% of a total cell suspension. Commercially available slide-based image-processing systems have been developed that provide sophisticated cell and tissue analysis functions using inexpensive personal-computer-based systems.

Antibodies↗

PubMed1987

Prognostic implications of proliferative activity and DNA aneuploidy in colonic adenocarcinomas.

Paraffin-embedded surgical specimens from 120 patients who underwent resections for primary untreated colonic adenocarcinoma were examined for proliferative activity, DNA aneuploidy, DNA index, and the proportion of aneuploid cells by flow cytometry. The results were correlated with survival times and clinical characteristics of the patients. The presence of metastases, both distant and restricted to local lymph nodes, was found to be a more potent adverse prognostic indicator than any DNA flow cytometry-derived parameter. Additional analyses were performed following stratification of patients into two groups on the basis of presence or absence of metastases. Analysis of 60 patients without metastatic involvement revealed a significant correlation between high proliferative activity, defined as more than 20% of cells in S-phase, and DNA aneuploidy. In fact, high proliferative activity was shown to be a more powerful adverse prognostic indicator in relation to survival than DNA aneuploidy in these cases after multivariate analysis. These results suggest that differences in proliferative activity may be an important biologic factor operative in the variable prognosis seen in colonic adenocarcinoma. In addition, they re-emphasize the importance of stratifying patients into groups based on metastatic involvement when evaluating other possible prognostic features in this disease.

Adenocarcinoma↗

PubMed1986

Assessment of cell cycle-associated antigen expression using multiparameter flow cytometry and antibody-acridine orange sequential staining.

A novel approach which enables direct assessment of the differential expression of cellular antigens in noncycling (G0) and cycling cell subpopulations is presented. The method involves flow cytometric analysis and sorting of cells stained by use of indirect immunofluorescence, followed by restaining using acid acridine orange, to relate the immunofluorescence of sorted lymphoid subpopulation(s) to cell proliferation status (i.e., G0 vs. G1 vs. S vs. G2 and M). In the present study, this technique successfully identifies the proliferation-associated modulation of a heterochromatin-associated antigen in pokeweed mitogen-stimulated human lymphoid cultures. The potential utility of this method for documenting early antigenic changes associated with the G0-G1 transition is discussed.

Acridine Orange↗

PubMed1986

Simultaneous nuclear antigen and DNA content quantitation using paraffin-embedded colonic tissue and multiparameter flow cytometry.

The simultaneous quantitation of nuclear antigens and DNA content is presented using monoclonal antibodies and flow cytometric analysis, with paraffin-embedded human colonic pathology specimens utilized as source material. The monoclonal antibodies evaluated were shown by immunogold electron microscopy to recognize nuclear proteins preferentially associated with interchromatin (p105) and heterochromatin (p34) regions. Indirect immunofluorescence analysis of p105 revealed two distinct G1-G0 cell subpopulations in cells from normal colonic epithelium and colonic adenocarcinomas. In addition, enhanced levels of both p105 and p34 were observed in aneuploid DNA content stemlines, relative to diploid cells. Cell-sorting experiments performed on cells sorted on the basis of p105 and DNA contents reveal the capability of this method for identifying morphologically heterogeneous cell subpopulations. Other data suggest that p105 is differentially expressed in well-differentiated versus poorly differentiated tumor regions. The potential utility of this approach for the retrospective study of proliferation-associated antigens and protooncogene protein products is discussed.

Adenocarcinoma↗

PubMed1986

Prognostic implications of ploidy and proliferative activity in diffuse large cell lymphomas.

Paraffin-embedded surgical biopsies from 50 patients with newly diagnosed diffuse large cell lymphoma (DLCL) were examined for proliferative activity and DNA aneuploidy by flow cytometry. These results were correlated with the clinical characteristics of these patients and the course of their disease. High proliferative activity, defined as less than 80% of cells in G0 or G1, was found to be the single most important pretreatment adverse prognostic factor in these patients. This relationship remained significant after correcting for poor performance status and advanced Ann Arbor stage, the other factors found to be associated with a shortened survival. DLCLs with high proliferative activity were more probable to present with extranodal involvement than those with lower proliferative activity. The mitotic count as determined by light microscopy did not correlate with flow cytometry-defined proliferative activity and may be a less accurate method for assessing this important biological characteristic in DLCL. DNA aneuploidy was detected in 62% of cases but did not appear to have any prognostic significance. Biopsies from patients who presented with lymphomatous bone marrow involvement, however, invariably demonstrated an aneuploid stemline. These results suggest that differences in proliferative activity may be an important biological basis for the variable prognosis seen in DLCL.

Adolescent↗

PubMed1986

Cell cycle changes and cytotoxicity in irradiated cultures of bovine aortic endothelial cells.

The purpose of this experiment was to determine the effect of ionizing radiation on cell number, lactate dehydrogenase (LDH) release, cell cycle distribution, [3H]thymidine incorporation, and autoradiographic labeling index in bovine aortic endothelial cells in vitro. Confluent endothelial monolayers were exposed to single doses of 0.5-10 Gy of 60Co gamma rays and were analyzed from 2 to 24 h postirradiation. Irradiated monolayers exhibited a time- and dose-dependent decrease in cell number, increase in LDH release, and redistribution of cells in the cell cycle. Cell cycle redistribution included an increase in the proportion of cells in S phase at 4 h after irradiation and a decrease in S phase at 24 h. The cells also exhibited a decrease in [3H]thymidine incorporation as early as 2 h after 5 Gy. This represented the most rapid radiation response observed in the present study. These data demonstrate that radiation cytotoxicity in confluent, plateau-phase endothelial monolayers is accompanied by changes in the cell cycle distribution of adherent cells, and that reduced [3H]thymidine incorporation is an early marker of radiation injury in this clinically important cell type.

Animals↗

PubMed1985

A method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry.

A preparative technique for the two-parameter flow cytometric study of nuclear antigen expression is reported. This method employs a brief sequential treatment of cells at 4 degrees C first with 0.5% paraformaldehyde and second with 0.1% Triton X-100 in phosphate-buffered saline followed by cellular staining with indirect immunofluorescence and propidium iodide. Using this technique, cellular morphology is preserved, cell clumping is minimized, and high-quality indirect immunofluorescence and DNA staining are obtained with a minimum of nonspecific labeling. Utilizing nuclear antigen-specific monoclonal antibodies in conjunction with this technique, the cell-cycle phase-dependent expression of such antigens is examined. From these data, the utility of two-parameter flow cytometry in the identification and quantification of cell-cycle-dependent modulation of nuclear antigens is discussed.

Antibodies, Monoclonal↗

PubMed1984

Improved detection of rare CALLA-positive cells in peripheral blood using multiparameter flow cytometry.

A major limitation to the detection of rare cell types in the peripheral blood using monoclonal antibodies is nonspecific binding of the antibody reagent to normal cells. Detection of rare common acute lymphoblastic leukemia antigen (CALLA)-positive cells in peripheral blood is significantly improved by using multiple flow cytometric parameters to exclude a variety of mature blood cells which may nonspecifically bind the antibody reagent. Monocytes and granulocytes are excluded by gating out cells with high 90 degrees light scatter. By gating on red fluorescence, a variety of mature cell types binding to phycoerythrin (PE)-conjugated Leu 3, Leu 2, and M3 monoclonal antibodies are also excluded. CALLA-positive lymphoblasts from 6 consecutive patients were not excluded on the basis of these parameters. Gating on log 90 degrees light scatter and log red fluorescence in this fashion reduced the incidence of nonspecific binding to peripheral blood mononuclear cells of a fluorescein-conjugated irrelevant monoclonal antibody by 98% from 308 cells per million to 5 cells per million. One CALLA-positive lymphoblast per 100,000 peripheral blood mononuclear cells could be detected in mixture experiments using this method. The normal range of CALLA-positive cells in adults is less than 16 cells per million peripheral blood mononuclear cells. This low background of CALLA-positive peripheral blood cells may permit the detection of early leukemic relapse in acute lymphoblastic leukemia by analysis of the peripheral blood. This methodology can be applied to the detection of any rare cell type by using phycoerythrin-conjugated antibodies to markers that the cell type does not possess.

Adult↗

PubMed1984

The effect in the KHT sarcoma of CCNU and MISO on cell cycle progression evaluated by flow-cytometry.

Previous studies using the KHT sarcoma have shown that misonidazole (MISO) enhances the cytotoxicity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) by as much as a factor of 2.0. In the present study flow cytometry was used to monitor the changing DNA distributions of cells dissociated from solid tumors at successive times following treatment with CCNU, applied either alone or in combination with 0.5 mg/g MISO. The proportion of cells in late S and the G2M phases of the cell cycle increased gradually after CCNU treatment. MISO did not significantly change this block in cell progression, which persisted for at least 48 hr after treatment in all cases. CCNU shows marked carbamoylating activity, which has been associated with inhibition of RNA processing and with the degree of chemopotentiation achieved with MISO. Consequently, to evaluate whether MISO chemopotentiation was influencing the RNA distributions in tumors, RNA histograms were generated using acridine orange to differentially stain cellular DNA and RNA. By 24 hr after treatment, CCNU clearly altered the distribution of RNA, but no significant differences could be detected between results obtained from drug and drug plus sensitizer treated groups. These studies demonstrate the effect of CCNU on cell cycle progression in vivo. The addition of MISO did not result in further perturbation of the total tumor population, suggesting that cell cycle redistribution does not play a major role in chemopotentiation by MISO.

Animals↗

PubMed1983

Long-term effects of activity and of calcium and phosphorus intake on bones and kidneys of female rats.

Over a 22-week period, young female rats in conventional cages gained significantly more weight than rats housed with free access to running wheels. A trend toward increased bone density was indicated in runners. In a second test, young female rats were housed under the same conditions for 30 weeks, but the active group was then forced to run in a treadmill for 30 minutes, 5 times a week, over a 43-week period. During this time rats were fed either diets providing adequate Ca (0.5%) and adequate P (0.4%) or high P (1.2%), or low Ca (0.3%) and high P (1.2%). Running appeared to benefit long bone density but could not completely compensate for a high P or a low Ca, high P intake. A bone that received only indirect stress during exercise (atlas) was not significantly affected by running. However, a high P diet significantly decreased atlas bone density, while the negative effect of this diet on the density of long bones was at a lower level of significance. A low Ca, high P diet clearly decreased bone density in all bones investigated. Running had no effect on kidney mineralization, but either a high P or low Ca, high P diet greatly increased the concentration of these minerals in the kidney.

Animals↗

PubMed1982

Isolation of quiescent cells from multicellular tumor spheroids using centrifugal elutriation.

A quiescent (nonproliferating) subpopulation was identified by flow cytometric analysis using two-step acridine orange staining in the EMT6/Rochester, N. Y. subline multicellular tumor spheroid, an in vitro culture system which provides a cellular microenvironment which mimics that of many of in vivo tumors. To isolate a viable quiescent cell subpopulation, centrifugal elutriation which allows for cell separation mainly on the basis of size was used. This technique provided single cells of relatively homogeneous cell volume which varied over a wide range (approximately 100 to 5000 cu microgram). Though the relatively small cell volume fractions were the most enriched (82%) in quiescent cells, such cells were also observed in significant numbers (congruent to 20%) even in the largest cell fractions. The cell clonogenicity of the various elutriation constant in fractions was also assessed and shown to be lowest (plating efficiency congruent to 20%) in the small spheroid cells but relatively constant in fractions containing intermediate and large cells (plating efficiency congruent to 50%). Continuous [3H]thymidine labeling indicated a slower rate of accumulation of labeled cells in the small spheroid cells, which may result from the transition of proliferating spheroid cells to the quiescent compartment during the course of labeling. These finding indicate the utility of centrifugal elutriation for quiescent cell characterization in in vitro tumor systems.

Animals↗

PubMed1981

Control of cellular proliferation in HeLa-S3 suspension cultures. Characterization of cultures utilizing acridine orange staining procedures.

Growth control is investigated in detail in fed and unfed HeLa-S3 suspension cultures. Two-step acridine orange staining and flow cytometric analysis indicated declines in cellular red fluorescence (proportional to RNA content) of 40-50% between exponential and plateau phase in both culture types. Cellular green fluorescence (DNA content) assessed simultaneously indicates an increment of cells with Gi-DNA content in plateau phase in the unfed cultures, while fed cultures show a brief increment in G1-phase cells in the transition phase followed by a recovery in plateau phase to a value similar to that of exponential cultures. Temporal declines in the 3H-thymidine pulse-labeling index are observed in both culture systems. These data along with the flow cytometry data indicate a distinct G1-arrest in the unfed plateau cultures and suggest a random arrest of cells about the cell cycle in fed plateau cultures. Acidic acridine orange staining and flow cytometric analysis furthermore indicate the occurrence of a quiescent population comprising approximately 345 of the total cells and consisting of both dead and viable cells in plateau phase unfed cultures. In contrast, fed plateau cultures show approximately 14% quiescent, mostly dead cells. Also, both culture systems show temporal declines in the clonogenic index and a longer cell-cycle transit time in plateau phase relative to exponential phase. These findings confirm earlier work which indicates that the environment has a profound influence on the mode of growth control for mammalian cells in vitro.

Acridine Orange↗

PubMed1980

Analytical cytometric approaches to heterogeneous cell populations in solid tumors: a review.

The problems encountered in studying the heterogeneity of cells in solid tumors is reviewed with emphasis on the role of various analytical cytometric assays for studying both the biology and the dynamics and proliferating, quiescent and dead malignant cells in vitro and in vivo. Due to advances in cytometric technology, many interesting in vitro studies on tumor cells heterogeneity have been and will be conducted over the next several years. For example, the acidic acridine orange staining of HeLa cells in suspension culture does readily discriminate between proliferating and quiescent cells. Some of these assays have been and others will be extended to in vivo studies. However, it is obvious that either the current analytical cytometric techniques must be modified and refined to permit better resolution for the complex situation in vivo or other new analytical cytometric techniques will have to be developed before many interesting studies on tumor cell heterogeneity in vivo can be addressed with reasonable efficiency.

Acridine Orange↗

PubMed1980

Total cellular RNA content: correlation between flow cytometry and ultraviolet spectroscopy.

Total RNA content in Chinese hamster ovary and HeLa-S3 cells determined by ultraviolet spectroscopy is compared with the red fluorescence distribution of acridine orange-stained cells observed by flow cytometry. A correlation coefficient of 0.93 is obtained when these methods of estimating RNA content are compared after various RNAse treatments. These data suggest that acridine orange staining effectively quantitates total cellular RNA content when analyzed by flow cytometry, although DNA is also shown to contribute a low but significant background of red fluorescence.

Acridine Orange↗