6 research outputs found
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Apoptosis (Programmed Cell Death) and the Evaluation of Chemosensitivity in Chronic Lymphocytic Leukemia and Lymphoma
Abstract 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
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Monoclonal Antibody to Single-Stranded DNA Is a Specific and Sensitive Cellular Marker of Apoptosis
The most widely used histochemical marker of apoptosis (in situend labeling, TUNEL) detects both apoptotic and necrotic cells and evaluates only late stages of apoptosis. Hence, a specific and sensitive cellular marker of apoptosis is needed to determine the role of apoptotic death in biology and pathology. The present study describes a novel immunohistochemical procedure for the staining of apoptotic cells using a monoclonal antibody (MAb) to single-stranded DNA. This MAb stained all cells with the morphology typical of apoptosis in etoposide-treated HL-60, MOLT-4, and R9 cell cultures, in which apoptosis was accompanied by high, moderate, and low levels of internucleosomal DNA fragmentation, respectively. TUNEL stained all apoptotic cells in HL-60 cultures, nearly 60% of apoptotic cells in MOLT-4 cultures, and only 14% of apoptotic cells in R9 cultures. Apoptotic R9 cells, which progressed into secondary necrosis, retained MAb staining and became TUNEL-positive. Necrotic cells in MOLT-4 cultures treated with sodium azide were stained by TUNEL, but were negative for MAb staining. All floating cells at a late stage of apoptosis in MDA-MB-468 cultures treated with cisplatin were stained by both MAb and TUNEL. However, among adherent cells in the early stages of apoptosis, MAb stained nearly 20 times more cells than TUNEL. In histological sections of human tumor xenografts, MAb detected clusters of apoptotic cells in viable tumor tissue, but did not stain cells in areas of central ischemic necrosis. In contrast, TUNEL stained nuclei in necrotic areas. Thus, MAb to single-stranded DNA is a specific and sensitive cellular marker of apoptosis, which differentiates between apoptosis and necrosis and detects cells in the early stages of apoptosis
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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 (1–5μM) 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 Ca
2+ 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
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\u27 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 Gw 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 (\u3c1 ± 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 histopathokxjy 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
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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