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K D Bauer

Publications and source records attributed to K D Bauer.

At least 19 recordsLinked to original sources
PubMed2000

Reliable and sensitive analysis of occult bone marrow metastases using automated cellular imaging.

The presence of occult bone marrow metastases (OM) has been reported to represent an important prognostic indicator for patients with operable breast cancer and other malignancies. Assaying for OM most commonly involves labor-intensive manual microscopic analysis. The present report examines the performance of a recently developed automated cellular image analysis system (ACIS; ChromaVision Medical Systems, Inc.) for identifying and enumerating OM in human breast cancer specimens. OM analysis was performed after immunocytochemical staining. Specimens used in this study consisted of normal bone marrow (n = 10), bone marrow spiked with carcinoma cells (n = 20), and bone marrow obtained from breast cancer patients (n = 39). The reproducibility of ACIS-assisted analysis for tumor cell detection was examined by having a pathologist evaluate montage images generated from multiple ACIS runs of five specimens. Independent ACIS-assisted analysis resulted in the detection of an identical number of tumor cells for each specimen in all instrument runs. Additional studies were performed to analyze OM from 39 breast cancer patients with two pathologists performing parallel analysis using either manual microscopy or ACIS-assisted analysis. In 17 of the 39 cases (44%), specimens were classified by the pathologist as positive for tumor cells after ACIS-assisted analysis, whereas the same pathologist failed to identify tumor cells on the same slides after analysis by manual microscopy. These studies indicate that the ACIS-assisted analysis provides excellent sensitivity and reproducibility for OM detection, relative to manual microscopy. Such performance may enable an improved approach for disease staging and stratifying patients for therapeutic intervention.

Bone Marrow Neoplasms↗
PubMed1998

BCL-2 expression correlates with lower proliferative activity in the intermediate- and high-grade non-Hodgkin's lymphomas: an Eastern Cooperative Oncology Group and Southwest Oncology Group cooperative laboratory study.

An inverse relationship between BCL-2 expression and cell cycle transition has been suggested by recent studies in murine models. To investigate the clinical relevance of these laboratory studies, a group of 116 paraffin-embedded non-Hodgkin's lymphoma (NHL) biopsy specimens (Working Formulation Groups D-H, and J) from a cooperative group study of cellular DNA content were analyzed for the 14;18 translocation using polymerase chain reaction (PCR)-based methods and, if sufficient tissue remained, for BCL-2 and BAX expression by immunohistochemistry. The results of these studies were then compared with the results of the previously performed flow cytometric analysis of ploidy and proliferative activity (S-phase-fraction). BCL-2 expression was inversely associated with proliferative activity (P = .001; n = 41), but there was no association between staining for Bax and %S-phase. Ploidy was not associated with either BCL-2 or BAX expression. The t(14;18) was detected in 21 of the 54 cases in which PCR-amplifiable DNA was recovered; 20 of these occurred at the major breakpoint region and 1 at the minor breakpoint region. High levels of BCL-2 or BAX expression occurred independently of t(14;18). There was no association between t(14;18) and either ploidy or proliferative activity. The inverse relationship between BCL-2 expression and proliferative activity in the intermediate- and high-grade NHLs is consistent with recent studies suggesting that Bcl-2 both retards entry into the cell cycle and inhibits apoptosis.

Cell Division↗
PubMed1998

Simultaneous measurement of cell cycle and apoptotic cell death.

Understanding the dynamics of cell death in conjunction with those of cell cycle can be illuminating in the investigation of various cellular behaviors. Robust assays for measuring such parameters are invaluable. Many assays of apoptosis and/or cell cycle use flow cytometry. This chapter describes two different assays to measure apoptosis and cell cycle simultaneously using flow cytometry. The first involves the use of terminal transferase (the "TUNEL" assay) together with propidium iodide for identification of cell cycle. The second uses fluorescently labeled annexin V, together with propidium iodide as an indicator of cell membrane integrity; and additionally Hoechst 33342 for determination of cell cycle. Each assay has positive and negative attributes. The terminal transferase assay is performed using fixed cells and is therefore useful in the analysis of samples collected over time. The annexin V assay is performed using unfixed cells, and thus provides information regarding membrane integrity. Other practical aspects of both assays are discussed.

Animals↗
PubMed1996

Apo-3, a new member of the tumor necrosis factor receptor family, contains a death domain and activates apoptosis and NF-kappa B.

BACKGROUND: Two receptors that contain the so-called "death domain' have been described to date: tumor necrosis factor receptor 1 (TNFR1) and Fas/Apo-1 (CD95); both belong to the TNFR gene family. The death domain of TNFR1 mediates the activation of programmed cell death (apoptosis) and of the transcription factor NF-kappa B, whereas the death domain of CD95 only appears to activate apoptosis. RESULTS: We have identified an additional member of the TNFR family, which we have named Apo-3. Apo-3 is a transmembrane protein of approximately 47 kDa that has similarity of members of the TNFR family in its extracellular, cysteine-rich domains. In addition, Apo-3 resembles TNFR1 and CD95 in that it contains a cytoplasmic death domain. The Apo-3 gene mapped to human chromosome 1p36.3, and Apo-3 mRNA was detected in several human tissues, including spleen, thymus, peripheral blood lymphocytes, small intestine and colon. Ectopic expression of Apo-3 in HEK293 or HeLa cells induced marked apoptosis. CrmA, a poxvirus inhibitor of Ced-3-like proteases which blocks death signaling by TNFR1 and CD95, inhibited Apo-3-induced apoptosis. Ectopic expression of Apo-3 also induced the activation of NF-kappa B. Apo-3 did not specifically bind to the Apo-2 ligand, suggesting the existence of a distinct ligand for Apo-3. CONCLUSIONS: These results identify Apo-3 as a third member of the TNFR family that activates apoptosis, and suggest that Apo-3, TNFR1 and CD95 engage a common apoptotic cell-death machinery. Apo-3 resembles TNFR1 because it can stimulate NF-kappa B activity and regulate apoptosis. Apo-3 mRNA is expressed in various tissues, consistent with the possibility that this receptor may regulate multiple signaling functions.

Amino Acid Sequence↗
PubMed1996

Prognostic implications of ploidy and proliferative activity in the diffuse, aggressive non-Hodgkin's lymphomas.

The International Index is a powerful predictor of outcome in the aggressive non-Hodgkin's lymphomas that is based solely on clinical features. Proliferative activity (% S-phase) measured by flow cytometry has been reported to have prognostic significance in many series and may represent a biologic correlate of clinical behavior that further defines prognosis. Flow cytometric analysis of cellular DNA content and proliferative activity (% S-phase) was performed on fixed paraffin-embedded biopsy specimens from 242 previously untreated patients with diffuse, aggressive non-Hodgkin's lymphomas entered on phase III intergroup clinical trials. The International Index was calculated for each patient based on stage, lactate dehydrogenase, performance status, number of extranodal sites, and age, as previously reported. The International Index consistently predicted response to therapy (P = .027) and survival (P = .007) in this series. DNA aneuploidy was shown in 57% of cases, but was not predictive of clinical outcome. The median % S-phase was 9.9 (median coefficient of variation, 3.6%), which was highly correlated with mitotic index (P = .0001). Although a trend associating low proliferative activity with good early survival and very high S-phase with a shortened survival was shown, International Index risk was the only significant predictor of survival in the multivariate analysis. Although proliferative activity quantitated by flow cytometric analysis of nuclei extracted from paraffin-embedded specimens is probably predictive of survival, it is a less powerful prognostic indicator than clinical parameters represented by the International Index and provides no additional prognostic information.

Adult↗
PubMed1996

Aequorea green fluorescent protein: simultaneous analysis of wild-type and blue-fluorescing mutant by flow cytometry.

Aequorea green fluorescent protein (GFP) has been expressed in a variety of cell lines and host organisms. A recent report (Heim et al.: Proc Natl Acad Sci USA 91:12501-12504, 1994) has documented that a GFP mutant with a single amino acid substitution (tyrosine 66 to histidine; Y66H-GFP) elicits altered spectral properties. Whereas wild-type GFP emits with a maximum at approximately 509 nm (green fluorescence), Y66H-GFP fluoresces with a maximum at approximately 448 nm (blue fluorescence). In this study we employed available argon and krypton ion laser lines to investigate the impact of laser excitation wavelength on the detection of Y66H-GEP by flow cytometry. Using transiently transfected 293 cells, a cellular subpopulation with elevated blue fluorescence was detectable with excitation at 407 nm, but not with ultraviolet (UV), 458 nm, or 488 nm excitation. The blue-fluorescing cells were further documented to express Y66H-GFP by immunoblot analysis of sorted cells. Finally, we demonstrated the simultaneous analysis of both wild-type and Y66H-GFP in cotransfected 293 cells using 407 nm excitation while collecting blue fluorescence at 460 +/- 20 nm (Y66H-GFP) and green fluorescence at 525 +/- 25 nm (wild-type GFP). These studies illustrate the potential for assessing differential gene expression by simultaneously analyzing multiple GFP species with multiparameter flow cytometry.

Cell Line↗
PubMed1996

Activation of apoptosis by Apo-2 ligand is independent of FADD but blocked by CrmA.

A new member of the tumor necrosis factor (TNF) cytokine family, designated Apo-2 ligand (Apo-21) [1] or TRAIL [2], has been shown recently to induce apoptosis in various tumor cell lines; however, its biological role is unknown. Here, we show that Apo-21, activated apoptosis in T-cell-enriched cultures of peripheral blood lymphocytes stimulated by interleukin-2 (IL-2), but not in unstimulated cells. This finding suggests that, like Fas/Apo-1 ligand and TNF [3-5], Apo-2L may play a role in regulating post-stimulation apoptosis of mature lymphocytes. Studies on the mechanism of Apo-2L action demonstrated marked membrane blebbing, a hallmark of apoptosis, within a few minutes of the addition of Apo-2L to tumor cells. Ectopic expression of a dominant negative mutant of FADD, a cytoplasmic protein that mediates death signalling by Fas/Apo-1 and by TNF receptor type 1 (TNFR1) [6-9], inhibited the induction of apoptosis by anti-Fas/Apo-1 antibody, but had little effect on Apo-2L function. In contrast, expression of CrmA, a cowpox virus-derived inhibitor of the Ced-2-like proteases ICE [10] and CPP32/Yama [11,12], blocked the induction of apoptosis by either Apo-2L or anti-Fas/Apo-1 antibody. These results suggest that Apo-2L activates a rapid, FADD-independent pathway to trigger a cell-death programme that requires the function of cysteine proteases such as ICE or CPP32/Yama.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Growth regulation of human breast and ovarian tumor cells by heregulin: Evidence for the requirement of ErbB2 as a critical component in mediating heregulin responsiveness.

Alterations in the expression of the epidermal growth factor (EGF) receptor ErbB family are frequently encountered in a number of human cancers. Two of these receptors, ErbB3 and ErbB4, are known to bind a family of related proteins termed heregulins (HRGs) or neu differentiation factors. In biologically relevant systems, interaction of HRG with ErbB3 or ErbB4 results in the transactivation of ErbB2. In this report, we show that ErbB2 is a critical component in mediating HRG responsiveness in a panel of human breast and ovarian tumor cell lines. Because HRGs have been reported to elicit diverse biological effects on cultured cells, including growth stimulation, growth inhibition, and induction of differentiation, we systematically examined the effect of rHRG beta 1 on tumor cell proliferation. HRG binding studies were performed with a panel of breast and ovarian tumor cell lines expressing a range of levels of ErbB2. The biological responses to HRG were also compared to EGF and to the growth-inhibitory anti-ErbB2 antibody, 4D5. In most cases, HRG stimulation of DNA synthesis correlated with positive effects on cell cycle progression and cell number and with enhancement of colony formation in soft agar. On each cell line tested, the HRG effects were distinguishable from EGF and 4D5. Our findings indicate that HRG induces cell proliferation in a number of tumor cell lines. In addition, we show that methods for measuring cell proliferation, as well as experimental conditions, are critical for determining HRGs effect on tumor cell growth in vitro.

Animals↗
PubMed1996

Heparin induces dimerization and confers proliferative activity onto the hepatocyte growth factor antagonists NK1 and NK2.

Hepatocyte growth factor (HGF) is a potent epithelial mitogen whose actions are mediated through its receptor, the proto-oncogene c-Met. Two truncated variants of HGF known as NK1 and NK2 have been reported to be competitive inhibitors of HGF binding to c-Met, and to function as HGF antagonists (Lokker, N.A., and P.J. Godowski. 1993. J. Biol. Chem. 268: 17145-17150; Chan, A.M., J.S. Rubin, D.P. Bottaro, D.W. Hirschfield, M. Chedid, and S.A. Aaronson. 1991. Science (Wash. DC). 254:1382-1387). We show here, however, that NK1 acts as a partial agonist in mink lung cells. Interestingly, NK1, which is an HGF antagonist in hepatocytes in normal conditions, was converted to a partial agonist by adding heparin to the culture medium. The interaction of NK1 and heparin was further studied in BaF3 cells, which express little or no cell surface heparan sulfate proteoglycans. In BaF3 cells transfected with a plasmid encoding human c-Met, heparin and NK1 synergized to stimulate DNA synthesis and cell proliferation. There was no effect of heparin on the IL-3 sensitivity of BaF3-hMet cells, and no effect of NK1 plus heparin in control BaF3 cells, indicating that the response was specific and mediated through c-Met. The naturally occurring HGF splice variant NK2 also stimulated DNA synthesis in mink lung cells and exerted a heparin-dependent effect on BaF3-hMet cells, but not on BaF3-neo cells. The activating effect of heparin was mimicked by a variety of sulfated glycosaminoglycans. Mechanistic studies revealed that heparin increased the binding of NK1 to BaF3-hMet cells, stabilized NK1, and induced dimerization of NK1. Based on these studies, we propose that the normal agonist activity of NK1 and NK2 in mink lung cells is due to an activating interaction with an endogenous glycosaminoglycan. Consistent with that model, a large portion of the NK1 binding to mink lung cells could be blocked by heparin. Moreover, a preparation of glycosaminoglycans from the surface of mink lung cells induced dimerization of NK1. These data show that the activity of NK1 and NK2 can be modulated by heparin and other related glycosaminoglycans to induce proliferation in cells expressing c-Met.

Animals↗
PubMed1995

Aequorea green fluorescent protein analysis by flow cytometry.

The isolation and expression of the cDNA for the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has highlighted its potential use as a marker for gene expression in a variety of cell types (Chalfie et al.: Science 263: 802-805, 1994). The longer wavelength peak (470 nm) of GFP's bimodal absorption spectrum better matches standard fluorescein filter sets; however, it has a considerably lower amplitude than the major absorption peak at 395. In an effort to increase the sensitivity of GFP with routinely available instrumentation, Heim et al. (Nature 373:663-664, 1995) have generated a GFP mutant (serine-65 to threonine; S65T-GFP) which possesses a single absorption peak centered at 490 nm. We have constructed this mutant in order to determine whether it or wild-type GFP (wt-GFP) afforded greater sensitivity when excited near their respective absorption maxima. Using the conventionally available 488 nm and ultraviolet (UV) laser lines from the argon ion laser as well as the 407 nm line from a krypton ion laser with enhanced violet emission, we were able to closely match the absorption maxima of both the S65T and wild-type forms of Aequorea GFP and analyze differences in fluorescence intensity of transiently transfected 293 cells with flow cytometry. The highest fluorescence signal was observed with 488 nm excitation of S65T-GFP relative to all other laser line/GFP pairs. The wt-GFP fluorescence intensity, in contrast, was significantly higher at 407 nm relative to either 488 nm or UV. These results were consistent with parallel spectrofluorometric analysis of the emission spectrum for wt-GFP and S65T-GFP. The relative contribution of cellular autofluorescence at each wavelength was also investigated and shown to be significantly reduced at 407 nm relative to either UV or 488 nm.

Base Sequence↗
PubMed1995

Effects of omeprazole on cell kinetics of carcinogen-induced colon tumours in rats.

This study was designed to evaluate the effects of hypergastrinaemia induced via suppression of gastric acid by omeprazole on carcinogen-induced colon cancer in rats. The carcinogen methylazoxymethanol (MAM), 30 mg/kg, was administered intraperitoneally at 6-weekly intervals to Sprague-Dawley rats. Four weeks after the last MAM injection, the first daily dose of omeprazole, 40 mg/kg, was given by gastric gavage to one group of rats, and the rest were given buffered methylcellulose vehicle. After 10 weeks of daily omeprazole or vehicle, the rats were anaesthetized with ether, blood samples obtained, and animals sacrificed. Gastrin levels in serum from omeprazole-treated rats were elevated nearly six-fold. DNA and RNA levels in gastric mucosa were unchanged by omeprazole, but protein content was somewhat reduced. No biochemical or histological changes related to omeprazole treatment were observed in normal colon. The number of tumours, tumour volumes, and total tumour burden were not significantly different in colons of vehicle- or omeprazole-treated rats. Analysis by flow cytometry revealed that the S phase fraction was lower in tumour cells from omeprazole-treated animals; and that the frequency of DNA aneuploidy was also reduced. The results indicate that while omeprazole-induced suppression of stomach acid in rats elevates levels of gastrin in serum, it does not substantially alter the biochemical or cellular characteristics of carcinogen-induced colon tumours.

Animals↗
PubMed1993

Growth factor receptor and related oncogene determination in mesenchymal tumors.

BACKGROUND: Sarcoma are rare malignant neoplasms that originate from a mesenchymal cell line. The epidermal growth factor receptor (EGF-R) has been identified in these malignant neoplasms by immunohistochemical techniques. METHODS: This investigation has evaluated the gene amplification and expression of EGF-R and the homologous oncogene c-erbB-2 in soft tissue and osseous sarcomas by Southern and northern blot analysis. RESULTS: Amplification of EGF-R and c-erbB-2 was identified in 2 of 117 (1.7%) and 6 of 105 (5.7%) of the sarcomas, respectively. Increased expression of EGF-R and c-erbB-2 was identified in 21 of 43 (49%) and 35 of 94 (37%) sarcomas, respectively. CONCLUSIONS: The expression of these two genes in sarcomas appears to occur independently and not be associated with tumor histologic characteristics, grade, size, DNA content, or proliferative activity.

Biomarkers, Tumor↗
PubMed1993

P-glycoprotein and alterations in the glutathione/glutathione-peroxidase cycle underlie doxorubicin resistance in HL-60-R, a subclone of the HL-60 human leukemia cell line.

HL-60-R, a multi-drug-resistant (MDR) subclone of the human leukemia cell line HL-60, was selected in continuous culture in doxorubicin (DOX) in the absence of mutagenic agents. When compared to the parent line HL-60, HL-60-R showed greater relative resistance to vinblastine than to etoposide, or to the selecting agent DOX. Co-exposure to verapamil, a known modulator of MDR, partially increased its sensitivity to DOX and vinblastine. The HL-60-R cell line stained positively with the P-glycoprotein-specific monoclonal antibody (MAb), C219, whereas the HL-60 parent was negative. Southern analysis showed 32-fold amplification of the mdrI gene in HL-60-R, whereas slot-blot analysis demonstrated 70-fold over-expression of the specific mdrI message in HL-60-R compared to HL-60. Northern blot analysis revealed the presence of 2 species of messenger RNA of sizes 5.1 kb and 4.5 kb. No transcripts were detectable in the parent. Flow cytometric analysis showed significantly reduced cellular retention of DOX as well as rapid efflux from the drug-resistant cell line. HL-60-R proved to be nearly 4 times more resistant to hydrogen peroxide than its parent, and 1,000 times more resistant to inhibition of cellular glutathione synthesis by D,L-buthionine sulfoximine (BSO). Verapamil modulated DOX resistance in HL-60-R incompletely but, in the presence of glutathione depletion, nearly completely reversed DOX resistance. Elevated levels of glutathione and glutathione-peroxidase activity were demonstrated, thereby implicating enhanced activity of the glutathione/glutathione-peroxidase cycle as an additional basis for its resistance to DOX. These findings suggest that an enhanced capacity for detoxifying oxyradicals may contribute to anthracycline resistance in acute leukemia.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Chromosomes 8, 12, and 17 copy number in Astler-Coller stage C colon cancer in relation to proliferative activity and DNA ploidy.

Fluorescence in situ hybridization using centromere-specific DNA probes to chromosomes 8, 12, and 17 was applied to 23 archival paraffin-embedded stage C colonic cancer specimens. Chromosome copy number was related to flow cytometric determinations of S-phase fraction and DNA ploidy. Three to eight copies of chromosomes 8, 12, and 17 were observed at mean frequencies of 28.7%, 37.8%, and 20.9%, respectively. The mean frequency of multiple copies of chromosome 12 was significantly greater than that for chromosome 17 (P < 0.0025). The mean frequency of single copies of chromosome 17 was significantly greater than that for chromosomes 8 and 12 (P < 0.0025 and P < 0.0005, respectively). Regarding the fourth quartile of cases, defined on the basis of the frequency of multiple chromosome copies, the proportion demonstrating moderate to high proliferative activity greatly exceeded the proportion displaying low proliferative activity. The same cases (most chromosomally aberrant) also generally demonstrated DNA aneuploidy. The results indicate a substantial degree of karyotypic instability in advanced colon cancer, particularly in cases with high proliferative activity and DNA aneuploidy.

Adenocarcinoma↗
PubMed1993

Evaluation of a dual-color flow cytometry immunophenotyping panel in a multicenter quality assurance program.

A basic immunophenotyping panel that employed dual-color combinations of fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated monoclonal antibodies (mAb; FITC-CD45/PE-CD14, FITC-IgG1/PE-IgG2, FITC-CD3/PE-CD8, FITC-CD3/PE-CD4, FITC-CD3/PE-CD16 + PE-CD56, and PE-CD19) was utilized in a quality assurance program to determine whether the 4 laboratories participating in a multicenter AIDS study obtained similar lymphocyte subset percentage values for T cells, B cells, NK cells, and CD4+ and CD8+ T cells. Over a 1 1/2 year period, 78 shared peripheral blood specimens were prepared and analyzed in each laboratory. The CD45bright CD14- percentage for each specimen was used to correct that individual's lymphocyte subset values. Interlaboratory coefficients of variation (CV) for the human immunodeficiency virus type I (HIV) seronegative (n = 38) and HIV-seropositive (n = 40) specimens using this panel were < 3% for total T cells; < 5% for CD4+ T cells and CD8+ T cells; < or = 17% for B and NK cells; and < 8% for CD4T/CD8T ratios. The 6-tube basic immunophenotyping panel has several notable features: a) for clinical studies, it permits comprehensive evaluation of an individual's major lymphocyte subsets, i.e., T, B, NK, and CD4+ and CD8+ T cells; b) for interlaboratory proficiency testing programs, it allows the detection of differences among laboratories in measurements of several functionally distinct cell populations; and c) for within-sample quality assurance, it provides several quality control checks, including the lymphosum, i.e., the sum of an individual's corrected T+B+NK values, a sum that was generally 100 +/- 5% on the HIV-seronegative specimens analyzed in this study.

AIDS Serodiagnosis↗