The number, distribution, and nucleating capacity of microtubule-organizing centers (MTOCs) has been investigated in a variety of cultured mammalian cells . Most interphase cells contain a single MTOC that is localized at the centrosome region and corresponds to the centriole and pericentriolar material . MTOCs, like centrioles, become duplicated during the S phase of the cell cycle and are equationally distributed to daughter cells in mitosis. Multiple MTOCs were rarely observed in cultured cells except in one cell line (neuroblastoma), which also displayed an equally large number of centrioles in the cytoplasm . The kinetics of microtubule assembly and the tubulin nucleating capacity of MTOCS was assayed by incubating tubulin-depleted, permeabilized 3T3 and simian virus 40-transformed 3T3 cells with phosphocellulose-purified 6S brain tubulin and microtubule assembly buffer . Initiation and assembly of 6S tubulin occurred in association with the cells' endogenous MTOCs, and the length, number, and distribution of microtubules generated about the organizing centers were regulated and cell specific . Our results are consistent with the notion that the specification of microtubule length, number, and spacial arrangement resides largely in the MTOCs and surrounding cytoplasm and not in the tubulin subunits .

Transformation of mammalian tissue culture cells by oncogenic viruses results in a 2-fold increase in the intracellular concentration of calmodulin quantitated by radioimmunoassay. The two pairs of companion cell lines used in this study were the Swiss mouse 3T3/simian virus 40-transformed 3T3 cells and the normal rat kidney (NRK)/Rous sarcoma virus-transformed NRK cells. The increased intracellular levels of calmodulin in the transformed cells are due to a greater increase in the rate of synthesis (3-fold) relative to the change in the rate ofdegradation (1.4-fold). On the other hand, no increases were observed in tubulin levels as quantitated by a colchicine-binding assay. The lack of change in tubulin concentration was accounted for by a 2-fold increase in the rate of degradation that is compensated by a similar increase in the rate of synthesis. The consequence of such changes in both transformed cell types is a 2-fold increase in the calmodulin-to-tubulin protein ratio relative to that in their nontransformed counterparts. 996The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Alpha transforming growth factors (alpha TGFs) were immunologically detected in the concentrated conditioned medium (CM) prepared from four human breast cancer cell lines and from primary cultures of human mammary epithelial cells, and in the tissue extracts prepared from normal, benign, and malignant breast biopsies. Immunoreactive alpha TGFs were quantitated by a competitive radioimmunoassay (RIA) using affinity-purified polyclonal sheep anti-rat alpha TGF antibodies which react with human alpha TGF but not with human epidermal growth factor (EGF). The relative level of RIA-detectable alpha TGFs in the CM from the breast cancer cell lines MCF-7, ZR-75-1, T47-D, and MDA-MB-231, and from the CM of primary cultures of human mammary epithelial cells, ranged from 0.02 to 0.85 ng/ml. MCF-7 or ZR-75-1 cells grown in the presence of 17 beta-estradiol (10(-8) M) for 48 h were found to release two- to three-fold more alpha TGFs into their CM than the same cells grown in the absence of estrogen. In detergent extracts prepared from normal breast tissue, a benign fibrocystic lesion, fibroadenomas and primary breast carcinomas, the relative alpha TGF concentrations were found to range from 1.5 to 6 ng/mg cell protein. No significant correlations were found between the alpha TGF levels and the pathological state of the tissues, the estrogen receptor status of the tumors, or the relative amounts of the ras gene protein p21ras in the tissues as determined by Western immunoblot analysis. The question of biological relevancy of alpha TGF for human mammary tumors will require further studies on synthesis and turnover of alpha TGF, the relationship between immunoreactivity and biological activity of alpha TGF, and differences in biological responsiveness of mammary tumor cells.

Monoclonal antibodies against the highly conserved ubiquitous calcium-binding protein, calmodulin (CAM), were produced by immunization of mouse primary spleen cell cultures. Dissociated spleen cells were cultured for 5 d in the presence of mixed thymocyte culture conditioned media (TCM) and purified bovine testes CaM (50 ng-1 mg). Following immunization, cells were fused with mouse myeloma cells (SP2/0, Ag 8.653) and cultured for 2-3 wk before initial screening for antibody. In five independent immunizations there was a range of 25-44% of the initial polyclonal cultures which produced antibodies reacting with purified CaM as determined by immunoassay. 80% of the cloned hybridoma produced IgM immunoglobulins while the remaining clones were IgG producers. This ratio was changed to 50% IgM and 50% IgG by subsequent extension of the in vitro immunization periods and reduced amounts of antigen and extended in vitro culturing. In vitro immunization introduces a new dimension to monoclonal antibody production where limited antigen or poorly antigenic proteins are of interest. The monoclonal antibodies produced in this study have enabled us to to selectively localize CaM in association with distinct subcellular structures, mitochondria, stress fibers, centrioles, and the mitotic spindle.