Abstract. We have explored the hypothesis that hypertrophy of vascular smooth muscle cells may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to serum mitogens by characterizing the effects of the potent growth inhibitor, transforming growth factor-[I (TGF-13), on both hyperplastic and hypertrophic growth of cultured rat aortic smooth muscle cells. TGF-13 inhibited seruminduced proliferation of rat aortic smooth muscle cells (EDs0 = 2 pM); this is consistent with previously reported observations in bovine aortic smooth muscle cells (Assoian et al. 1982. J. Biol. Chem. 258:7155-7160). Growth inhibition was due in part to a greater than twofold increase in the cell cycle transit time in cells that continued to proliferate in the presence of TGF-13. TGF-I~ concurrently induced cellular hypertrophy as assessed by flow cytometric analysis of cellular protein content (47 % increase) and forward angle light scatter (32-50% increase), an index of cell size. In addition to being time and concentration dependent, this hypertrophy was reversible. Simultaneous flow cytometric evaluation of forward angle light scatter and cellular DNA content demonstrated that TGF-I~-induced hypertrophy was not dependent on withdrawal of cells from the cell cycle nor was it dependent on growth arrest of cells at a particular point in the cell cycle in that both cycling cells in the G2 phase of the cell cycle and those in Gm were hypertrophied with respect to the corresponding ceils in vehicle-treated controls. Chronic treatment with TGF-I~ (100 pM, 9 d) was associated with accumulation of cells in the G2 phase of the cell cycle in the virtual absence of cells in S phase, whereas subsequent removal of TGF-I~ from these cultures was associated with the appearance of a significant fraction of cycling cells with >4c DNA content, consistent with development of tetraploidy. Results of these studies support a role for TGF-I~ in the control of smooth muscle cell growth and suggest that at least one mechanism whereby hypertrophy and hyperploidy may occur in this, as well as other cell types, is by alterations in the response to serum mitogens by potent growth inhibitors such as TGF-I~.C ELLULAR enlargement or hypertrophy plays a prominent role in the postnatal growth of many tissues, as well as in physiological and pathological hypertrophy of a variety of tissues (4). However, relatively little is known regarding the mechanisms that control cell size. Even less is known concerning the control mechanisms for the DNA endoreduplication and polyploidy that often accompanies cellular hypertrophy, although its occurrence is widespread in eukaryotic cells in vivo, occurring in terminally differentiated cardiac myocytes (14,15,29), and neurons (22), as well as in nonterminally differentiated hepatocytes (8), smooth muscle cells (10,23,30), and other cell types (see review by Brodsky and Uryvaeva [8]).

Murine B cell lines such as WEHI-231, BAL17 and M12.4.1 are frequently used as model systems to study signal transduction, cell cycle regulation, and apoptosis. Dissection of these processes often involves expressing exogenous genes in these cells. Electroporation is an inefficient method to express genes in B cell lines and requires several weeks to isolate and analyze clones, followed by an additional one to two weeks to grow sufficient cells for biochemical experiments (e.g. immunoprecipitations). In this report, we describe an optimized procedure for retroviral-mediated gene transfer into murine B cell lines that allows one to obtain a pure population of cells expressing an exogenous gene within 4 days. Two days post-infection, between 10% (BAL17 and M12.4.1 cells) and 70% (WEHI-231 cells) of the cells express the exogenous gene. Culturing the cells for an additional 48 hours with puromycin kills all the non-infected cells and yields a pure population of cells that express the exogenous gene. Sufficient cells for biochemical experiments can be obtained by expanding the cell culture for an additional 5 to 7 days. This rapid and efficient retroviral-mediated gene transfer procedure can greatly expedite the study of signal transduction and other processes in B cells.