Abstract 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.
Abstract 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 mdr I gene in HL‐60‐R, whereas slot‐blot analysis demonstrated 70‐fold over‐expression of the specific mdr I 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 sulfoxirnine (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 glutathionel/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.
Circulating endothelial cells (CECs) are increased in sickle cell disease, myocardial infarction, and acute lung injury. The purpose of this study was to determine whether CECs are a prognosticating marker for the development of pneumonia in burn patients with/without inhalation injury in addition to their relationship to proinflammatory cytokines. There were 24 patients: 6 with inhalation injury, 5 with burn only,and 13 with burn plus inhalation injury. CECs were measured by anchored cytometry (Clarient ChromaVision, San Juan Capistrano, CA). In addition, plasma levels of tumor necrosis factor-alpha, interferon-gamma, and interleukins (IL)-10, IL-6, IL-4, and IL-2 were compared with CEC levels. Patients with inhalation injury had a significant (P < .001) paucity of CECs compared with the thermally injured with inhalation. There was a statistically significant increase in inteferon-gamma, tumor necrosis factor-alpha, and IL-6, IL-4, and IL-2 compared with control patients (P < .01), with a concomitant increase in the number of CECs. The numbers of CEC levels did not prognosticate which patients would develop pneumonia. Burn patients with/without inhalation injury had concurrent increase in CECs and proinflammatory cytokines during the acute phase of injury.
Abstract 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 of proliferating, quiescent and dead malignant cells in vitro and in vivo . Due to advances in cytometric technology, many interesting in vitro studies on tumor cell 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.
To define the role of flow cytometry as a prognostic indicator in early cancers of the uterine cervix.Flow cytometry was used to determine ploidy, DNA index, and S-phase fraction on 141 samples from the tumors of 53 women with stage IB cancers of the cervix treated by radical hysterectomy and pelvic lymphadenectomy as primary therapy. Multiple samples of the same tumor were analyzable for 47 (89%) of the subjects. One-way analysis of variance for the multiple samples was used to compare the heterogeneity of flow cytometry data, both within each tumor and between patients. Flow cytometry results, as well as previously described clinical and pathologic prognostic factors, were correlated to recurrence and survival using Cox regression hazard ratios and Kaplan-Meier estimates.We found DNA aneuploidy in 25 (47%) of the cancers, with a mean (+/- standard error) DNA index of 1.52 +/- 0.07. The mean S-phase fraction was 7.6 +/- 0.4% for diploid tumors and 9.2 +/- 0.4% for aneuploid tumors. The cancers from 24 women (45%) were homogeneously diploid, 18 (34%) were consistently aneuploid, and five (9%) had mixed diploid/aneuploid samples. Analysis of variance of the multiple samples for each woman revealed a greater standard deviation (SD) between patients than within any individual tumor for both DNA index (ratio of between SD to within SD 2.1; P < .0001) and S-phase fraction (ratio 1.6; P < .0001). Of the previously described clinical and pathologic prognostic factors, only depth of invasion, expressed as either percent of cervical wall thickness or as thirds, was correlated with recurrence or survival. Neither the DNA index nor S-phase fraction correlated significantly with recurrence or survival.These results suggest that alterations in the DNA content or proliferative activity of early invasive cancers of the uterine cervix do not reflect biologic behavior in terms of recurrence or survival, and that this behavior is not due to tumor heterogeneity.
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