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Biomedical subjects

E V Sugarbaker

Publications and source records attributed to E V Sugarbaker.

At least 19 recordsLinked to original sources
PubMed1995

Synergistic induction of apoptosis in breast cancer cells by tamoxifen and calmodulin inhibitors.

Breast cancer cells are relatively resistant to the induction of apoptosis (AP) and drug regimens which readily activate apoptotic death, may enhance the antitumor effect. Rapid and intensive induction of apoptosis was observed in estrogen receptor positive and negative breast cancer cell cultures treated with tamoxifen (TMX) combined with the calmodulin antagonists trifluoperazine (TFP) or W7. TMX (1-5 microM) alone or calmodulin antagonists alone did not induce apoptosis. Importantly, intensive apoptosis was also induced by TMX and TFP in the cells obtained from primary human breast carcinomas. Inhibition of the Ca2+ calmodulin signaling pathway is an effective way to activate apoptotic death in epithelial cells. Combination of TMX with non-toxic calmodulin inhibitors may increase the preventive and therapeutic effects of TMX.

Apoptosis↗
PubMed1994

Pleiotropic drug resistance and survival advantage in leukemic cells with diminished apoptotic response.

Cell line R9 generated by continuous exposure of MOLT-4 cells to adriamycin was cross-resistant to a variety of unrelated drugs. The following data indicate that diminished apoptotic response was the mechanism of acquired pleiotropic drug resistance: (i) apoptosis was a common mechanism of cell death for agents expressing cross-resistance; (ii) induction of apoptosis by drugs, medium depletion and serum deprivation was decreased in R9 cells; (iii) DNA degradation in apoptotic cells was lower in resistant lines, probably reflecting a modification of apoptotic pathway in resistant cells; (iv) inhibition of cell division and DNA synthesis by drugs was similar in sensitive and resistant cells. These data indicated a similar level of initial damage, as typical for resistance based on modified apoptotic response. There was no difference in bcl-2 protein level between sensitive and resistant cells. Thus acquired pleiotropic resistance and diminished apoptotic response in R9 cells were induced by a bcl-2-independent mechanism. Surface T-cell antigen CD4 was expressed in MOLT-4 and lost in R9 cells. The role of CD4 down-regulation in apoptosis-related drug resistance remains to be explored. The association between acquired pleiotropic drug resistance and increased survival capacity in unfavorable growth conditions indicated that drug-induced selection of cells with diminished apoptotic response may stimulate neoplastic progression. Alkylating agents induced similar cytotoxicity and only slightly lower apoptosis in R9 cells in comparison with MOLT-4 cells. Our data show that some drugs may overcome acquired pleiotropic drug resistance based on the modified apoptotic pathway.

Antineoplastic Agents↗
PubMed1993

Apoptosis (programmed cell death) and the evaluation of chemosensitivity in chronic lymphocytic leukemia and lymphoma.

Chronic lymphocytic leukemia and lymphoma cells were treated with antitumor drugs in vitro and analyzed by flow cytometry to measure the number of apoptotic (AP) cells and DNA damage in the cells that escaped apoptotic death. AP cells were identified by a high sensitivity of DNA to thermal denaturation, which induced binding of antibody to single-stranded DNA, and by decreased stainability of cells with the intercalating DNA dye propidium iodide. The appearance of AP cells was prevented by Zn++ and inhibited by phorbol ester. AP cells were induced by alkylating agents, antimetabolites, and anthracyclines. A linear relationship between L-phenylalanine mustard dose and the number of AP cells was observed. A synergistic interaction between drugs was detected by an increased number of AP cells and by the intensity of DNA damage in non-apoptotic cells. A most interesting example of synergism was the combination of alkylating agents with fludarabine. Linearity of dose-response curves, and the capability to detect drug synergism and to evaluate variable response of cells from different patients to single agents and combinations suggest that flow cytometry of apoptosis will provide a basis for chemosensitivity tests in leukemia and lymphoma.

Antineoplastic Agents↗
PubMed1991

Intercellular transfer of drug resistance.

The effect of L-phenylalanine mustard (L-PAM) on heterogeneous cell populations containing sensitive and resistant cells was evaluated by flow cytometric analysis of DNA damage. Cell cultures were treated with L-PAM for 1 h, fixed, and stained with anti-DNA monoclonal antibody which detects DNA damage induced by alkylating agents. DNA damage was significantly lower in sensitive A2780 cells cocultured with resistant A549 or A2780/PAM cells than in A2780 cells grown separately. Decrease of DNA damage in sensitive cells did not occur when sensitive and resistant cells were grown in common medium without direct contact. Transfer of drug resistance in cocultures was prevented by phorbol ester which is known to inhibit metabolic cooperation via cell junctions. Treatment of cocultures with buthionine sulfoximine increased DNA damage in resistant cells and prevented decrease of DNA damage in sensitive cells. Glutathione (GSH) content in A2780 cells cocultured with A549 cells was significantly higher than GSH content in A2780 cells grown separately. We conclude that decreased response of sensitive cells in cocultures was induced by contact transfer of GSH from GSH-rich resistant cells to sensitive cells. Intercellular transfer of drug resistance demonstrated by analysis of DNA damage was confirmed by colony formation assay. Treatment with L-PAM and Adriamycin killed all cells in A2780/MDR and A549 cultures. Coculture of these lines survived combination treatment because transfer of GSH to multidrug-resistant cells from GSH-rich A549 cells induced resistance to L-PAM and Adriamycin in a single cell. The presence of 2% A549 cells increased resistance of A2780/MDR cells to L-PAM. Phorbol ester eliminated resistance of coculture to combination treatment. Metabolic cooperation between cell subsets with different mechanisms of drug resistance induced resistance to treatment with drugs of different classes (multiclass drug resistance). Inhibition of cell cooperation may improve the response of tumors to combination chemotherapy.

Antimetabolites↗
PubMed1990

Flow cytometric analysis of DNA damage and repair in the cells resistant to alkylating agents.

DNA damage in the cells sensitive and resistant to alkylating agents was determined by flow cytometry analysis of cells stained with anti-DNA monoclonal antibody (MOAB) F7-26. MOAB F7-26 interacted with single-stranded regions in alkylated DNA, and the binding of antibody to the cells increased in proportion to the decrease in cell viability. Development of resistance to L-phenylalanine mustard (L-PAM) in A2780 cells was associated with decreased immunoreactivity of DNA with MOAB F7-26. Fluorescence was significantly lower in resistant cells than in sensitive cells, and the difference in the binding of MOAB between two cell types increased with the dose of L-PAM. The enhancement of L-PAM cytotoxicity to resistant cells by buthionine sulfoximine and hyperthermia was accompanied by a proportional increase of MOAB F7-26 binding to DNA. The same relative potential of sensitization regimens was established by cell survival and MOAB staining. The time course of DNA repair established by decrease of MOAB binding after L-PAM removal was similar in sensitive and resistant cells. Resistance of A2780 cells to L-PAM was associated with low initial level of DNA damage and with decreased cytotoxicity per unit of damage. We conclude that resistant cells could be distinguished from sensitive cells by staining with MOAB F7-26 and that the sensitization of resistant cells could be quantitatively predicted by flow cytometry analysis of MOAB binding.

Alkylating Agents↗
PubMed1990

Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage.

DNA damage was measured by flow cytometric analysis of cells sensitive and resistant to alkylating agents. Human ovarian carcinoma cell line A2780 and a subline which is 7 times more resistant to L-phenylalanine mustard (L-PAM) were treated with the drug, fixed, and stained with monoclonal antibody (MOAB) F7-26 which detects single-stranded regions in alkylated DNA. Mean fluorescent intensity was measured on a flow cytometer. Cells were heated before staining to amplify single-strandedness in alkylated DNA. Significantly larger amount of MOAB was bound to DNA in sensitive than in resistant cells. Fluorescence increased by 80 channels per micrograms L-PAM insensitive cells and only by 17 channels in resistant cells. Sensitive and resistant cells were treated with L-PAM, mixed in different proportions, and stained with MOAB. Populations of sensitive and resistant cells were clearly separated on fluorescence histograms by more than a decade difference in fluorescence intensity. Presence of 2-5% resistant cells was detected among sensitive cells as a separate cell subset. We conclude that staining with MOAB F7-26 can be used as an indicator of cell sensitivity or resistance to alkylating agents. Detection of minor subsets of resistant cells in heterogeneous populations by FCM analysis may be useful for monitoring emerging drug resistance.

Alkylating Agents↗
PubMed1984

The effects of syngeneic soluble tumor membrane extract on concomitant spleen cell immunity and spontaneous metastases.

The effects of a soluble tumor KCl extract, containing membrane antigen on spontaneous metastases and spleen cell immunity were studied in a syngeneic C57BL/6J murine sarcoma model. The extract was shown to be antigenically distinct from normal tissue by tumor immunization rejection experiments. Tumor soluble extract (SE) was administered daily during the growth of a murine sarcoma. The afferent and efferent arc of immunity were monitored by proliferative index (PI) and in vitro cytotoxicity of spleen cells, as well as the incidence of metastases. PI was significantly activated in the group receiving sarcoma SE as compared to the two control groups receiving muscle SE or saline P less than 0.05. However, in vitro cytotoxicity was significantly depressed on Days 7 and 14 (P less than 0.05) of tumor growth in the mice receiving sarcoma SE. The incidence of metastases was significantly increased to 70% in the sarcoma SE group as compared to the incidence in the control groups of 50% P less than 0.05. This data supports the hypothesis that release of soluble antigen membrane components by growing tumor facilitates the growth of metastases in this model.

Animals↗
PubMed1984

Dual immunofluorescent analysis of human peripheral blood lymphocytes.

These studies reveal that a dual immunofluorescent labeling method is useful for enumerating cells from human peripheral blood that bear the helper, suppressor, and/or T-cell receptors. Fluorescein (FL)-conjugated Leu-3a + 3b antibodies were used to enumerate Helper (H) T-lymphocytes, while the B-phycoerythrin (B-PE)-conjugated Leu-2a antibodies were utilized for quantitating suppressor (S) T-lymphocytes. FL-conjugated Leu-4 antibodies were used to measure the T-lymphocyte activity. Dual immunofluorescent stained lymphocytes, prepared either from whole blood or by Ficoll-Hypaque, gradient cell separation, were analyzed by flow cytometry. Two light scatter parameters, forward and 90 degree, were used to define the lysed erythrocyte, lymphocyte, monocyte, and granulocyte populations. Only the lymphocytes were analyzed for dual immunofluorescence activity. The helper, suppressor, and T-lymphocyte distributions from 100 controls were as follows: The average percentages +/- SD of the helper and suppressor cells were 41.2 +/- 7.2 and 23.0 +/- 7.2, respectively. The H/S ratio was 1.99 +/- 0.77, while the T-cell distribution on 28 patients was 71.4 +/- 7.7. The Ficoll-Hypaque purified lymphocytes and lysed whole blood lymphocytes compared favorably in their H/S ratios. A comparison was made between the percentages of helper and suppressor cells enumerated by fluorescent microscopy and flow cytometry in which correlation coefficients of 0.80 and 0.86 were determined, respectively. These studies show that helper and suppressor T-lymphocytes can be quantitated simultaneously by flow cytometry, which enables one to correlate the phenotypic activities of two antibodies against cell surface receptors and permits the measurement of a large number of samples in a minimal time.

Adult↗
PubMed1984

Prognostic indicators including DNA histogram type, receptor content, and staging related to human breast cancer patient survival.

Primary tumors from breast cancer patients were evaluated for the biochemical presence of three steroid cytosolic receptors and by DNA histogram analysis using flow cytometry. These parameters were compared with the histological and staging diagnoses and the patients' survival over a 36-month period. A total of 74 patients with primary breast tumors were evaluated. The breast samples invariably demonstrated a peak population of diploid G0/1 cells which contained 2C amounts of DNA, as determined by mixing experiments using normal human breast tissues or trout erythrocytes as fixed standards. The tumors were classified into five DNA histogram types based on their DNA index distributions established by flow cytometry. These results showed that 21% of the tumors were diploid and indistinguishable from the diploid population of normal breast cells, 8% were hypodiploid, 11% were hypertetraploid, 8% were multiploid, and the remaining 52% were hyperdiploid. The DNA index values varied from 0.78 (hypodiploid) to 2.60 (hypertetraploid). The percentages of S-phase cells were lowest in the diploid and hypertetraploid tumors and highest in the hypodiploid tumors. Among the 24 patients who died during the 36-month follow-up, 92% (22 of 24) were classified in one of the aneuploid groups. Three high-risk groups identified on the basis of survival after 36 months were distinguished: hypodiploid (50% survival); multiploid (43% survival); and hyperdiploid (50% survival). Rates of survival in the diploid and hyperdiploid groups were 87 and 71%, respectively. The hypodiploid group was distinguished by having the lowest mean estrogen cytosolic receptor value [26 +/- 13 (S.D.) fmol/mg], progesterone cytosolic receptor value (13 +/- 15 fmol/ mg), and androgen cytosolic receptor value (less than 1 +/- 1 fmol/mg). In contrast, the diploid tumors had some of the highest receptor values, with mean estrogen cytosolic receptor value equal to 102 +/- 114 fmol/mg, progesterone cytosolic receptor value equal to 74 +/- 110 fmol/mg, and androgen cytosolic receptor value equal to 65 +/- 80 fmol/mg. The lowest survival rates (17% after 36 months) occurred in patients over 67 years of age who had aneuploid tumors, compared to 100% survival in patients over 67 years of age with diploid tumors. Our results demonstrate the value of using flow cytometry and steroid receptor values as supplements to histopathology for the characterization of subgroups of mammary cancer patients. The ability to identify patients with a good prognosis compared to those at high risk of recurrence and death will be valuable in the design of future prospective treatment studies.

Aneuploidy↗
PubMed1983

DNA flow cytometry of pleural effusions. Comparison with pathology for the diagnosis of malignancy.

A study was undertaken to determine the potential value of DNA flow cytometry (FCM) for the diagnosis of malignancy in pleural effusions and to compare its results with those of traditional cytomorphology. Forty-one pleural fluids from 37 patients were evaluated by DNA FCM and routine cytologic techniques. Of the 41 pleural fluids, 29 (70.7%) demonstrated an abnormal DNA content by FCM. Two of the 29 pleural fluids had been originally diagnosed as benign by cytology. Cytologic review of these two cases showed no abnormal cells; therefore, there were no false-negative cases based on cytology. In 12 (29.3%) of the 41 fluids, DNA FCM and cytologic evaluation both indicated benign processes. Our preliminary observations indicate that FCM is an accurate and reliable technique that may be of aid in the diagnosis of malignant effusions. The technique may prove to be of special value in the differential diagnosis of reactive mesothelial cells versus malignant mesothelioma as well as for following patients who receive chemotherapy for malignant pleural effusions. DNA FCM may also complement cytomorphologic diagnoses in other serous, exfoliative or aspiration material.

Chromosome Aberrations↗
PubMed1981

The APUD system and its apudomas.

As sensitive radioimmunoassays for the detection of polypeptide hormones are developed, the exciting discovery of a diffusely distributed system of interrelated endocrine cells has begun a new era of endocrinology. This system, although anatomically disassociated, is bound together by a number of common features such as its biosynthetic mechanism, histochemical and ultrastructural features, and embryologic origin (Table I). The most prominent feature, however, is their biosynthetic pathways for hormone production, from which the acronym APUD has been derived. These are the capacity for Amine Precursor Uptake such as DOPA and then subsequent Decarboxylation, resulting in the synthesis of bioactive amines or polypeptide hormones. Hyperplasias or neoplasms of these cells are defined as apudomas. In the last ten years a great deal of research has rapidly altered the original concepts of this system, especially in terms of its embryologic origin, physiologic interrelationships, classification, as well as the addition of many new APUD cell members. These will be reviewed, and the origin, diagnosis, and treatment of each recognized apudoma will be synthesized in light of its membership within the APUD system.

APUD Cells↗
PubMed1980

Mechanisms of levamisole-induced granulocytopenia in breast cancer patients.

Five of 39 (13%) women treated with adjuvant combination chemotherapy plus levamisole immunotherapy after mastectomy for Stage II or III breast cancer developed levamisole-induced granulocytopenia. This complication occurred in each of the women between six and ten weeks after the completion of six months of combination chemoimmunotherapy when they were taking levamisole alone. Although none of the patients had an HLA B-27 locus and leukoagglutinins could not be demonstrated, complement-dependent, IgM mediated, peripheral destruction of granulocytes was documented using a microgranulocytotoxicity assay. In addition, a factor(s) present in serum from patients developing levamisole-induced granulocytopenia caused suppression of bone marrow granulocyte progenitor cells (CFU-C). The possible relationships between levamisole-induced peripheral granulocyte destruction and bone marrow CFU-C suppression are discussed.

Acute Disease↗
PubMed1980

Preparation of tissues for DNA flow cytometric analysis.

A method for measuring DNA in tissue cells by flow cytometry utilizing a one step combination nuclear isolation-DNA fluorochrome staining procedure is described. A variety of cells and tissues, both in vivo and in vitro, was used to illustrate the universal nature of this technique. These included murine bone marrow, liver testicle, sarcoma brain tumor, rat pancreatic islets, human peripheral blood, colon mucosa, colon cancer, sarcoma and brain tumor tissues. A special nuclear isolation medium, which contained either of the DNA fluorochromes, 4',6-diamidino-2 phenylindole-2 HCl or propidium iodide, was utilized successfully to isolate single suspensions of DNA fluorochrome stained nuclei in a rapid (5-10 min), consistent manner from a variety of tissues and cells. Multiple sampling of the same tissue or comparison between whole tissues and their single cell isolates showed that a representative sample was being obtained.

Amidines↗