Analysis of the stimulation-inhibition paradox exhibited by lymphocytes exposed to concanavalin A.

We have previously reported that agents that disrupt microtubules, such as colchicine, inhibit the growth stimulation of lymphocytes and arrested fibroblasts; other workers have recently reported enhanced stimulation of fibroblasts in the presence of the same drugs. In the present studies, we resolve this conflict by demonstrating that the stimulatory and inhibitory effects of microtubule disruption depend upon the density and the cell type of the treated cultures. Our analysis included an examination of three variables: (i) cell density (sparse or confluent), (ih) cell type (resting fibroblasts from mouse or chicken embryos or from the permanent 3T3 mouse fibroblast line), and (iii) treatment with colchicine and related drugs in the presence or absence of various mitogens such as serum, insulin, and epidermal growth factor. We found that colchicine augmented mitogenesis in confluent cultures of all cell types.Colchicine by itself appeared to be mitogenic only for confluent chicken embryo fibroblasts. In sparse cultures with minimal cell-cell contacts, however, there were differences between embryonic cells and the 3T3 cell line. In confirmation of our revious reports, disruption of microtubules by colchicine inhibited the mitogenic stimulation of sparse cultures of embryonic chicken or mouse fibroblasts. In contrast, fibroblasts of the permanent 3T3 line in sparse cultures were stimulated by some mitogens despite the presence of colchicine. The augmentative effects of coichicine on the stimulation of confluent cultures were synergistic with the mitogens, and colchicine allowed response to otherwise submitogenic doses of growth factors. Kinetic studies indicated that the stimulatory and inhibitory effects of colchicine are separable and that both can operate simultaneously. The experiments suggest that the regulation of growth by nutrient deprivation and the regulation by density dependence proceed at least in part by different mechanisms.

mentioning

confidence: 99%

mentioning

confidence: 99%

Density-dependent stimulation and inhibition of cell growth by agents that disrupt microtubules

McClain

Edelman

1980

“…Colchicine appears to block this early recruitment event (3). High doses of Con A induce anchorage modulation of surface receptors and inhibit mitogenesis but do not interfere with the primary growth signal (5).…”

mentioning

confidence: 99%

Role of surface modulating assemblies in growth control of normal and transformed fibroblasts.

McClain

D’Eustachio

Edelman

1977

Self Cite

Cellular microtubules, microfilaments, and surface receptors have been postulated to form a surface modulating assembly that regulates surface receptor mobility and cell growth. To test this hypothesis, we examined three agents known to affect cell growth [colchicine, concanavalin A (Con A), and the src gene product of Rous sarcoma virus] for their effects on chick embryo fibroblasts. Individual cells from serum-starved normal fibroblast populations became committed to enter S phase at various times over a 12 hr period after exposure to serum. Colchicine and other microtubule-disrupting agents blocked entry into S phase at a point close to the commitinent point for each cell. The lectin Con A also blocked entry into the S phase when present in doses sufficient to modulate surface receptor mobility. In contrast, succinyl-Con A, which does not induce surface modulation, had no effect. Both Con A and colchicine blocked the appearance of cytopsmic factors capable of stimulating DNA replication in a cellfr system.To study endogenous effects on the surface modulating assembly, we infected fibroblasts with a Rous sarcoma virus (thNY68) having a temperature-sensitive mutation in the transforming (src) gene. We have previously shown that microtubular and microfilamentous structures of the surface modulating assembly are direct or indirect targets of the src gene product with consequent reduction in the capacity of Con A to induce surface modulation. TsNY68-infected fibroblasts shifted to the nonpermissive temperature acquired normal microtubular morpholoky more rapidly (2 hr) than cells grown at the permissive temperature in the presence of protein synthesis inhibitors (7.5 hr) This suggests that the src gene product acts directly on the surface modulating assembly rather than via the nucleus or at

“…In an earlier report, McClain and Edelman (19) suggested that lymphocytes were responsive to high doses of Con Proc. Natl.…”

Section: Discussionmentioning

confidence: 99%

Activation of early enzyme production in small lymphocytes in response to high, nonmitogenic concentrations of concanavalin A

Degen

Morris

1980

Lymphocyte mitogenesis is generally assessed by measuring the incorporation of [3Hlthymidine into DNA. By this criterion, small lymphocytes, which are activated by relatively low doses of concanavalin A, are either unresponsive to or inhibited by higher concentrations. Because lymphocytes begin to synthesize DNA about 24 hr after addition of mitogen, the response is far removed temporally from the initial stimulus. We have chosen to use the induction of S-adenosylmethionine decarboxylase (Sadenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) to assess early activation events in bovine lymphocytes. Adenosylmethionine decarboxylase induction is bimodal, with an initial phase beginning 3 hr after addition of concanavalin A and a second wave coinciding with the onset of DNA synthesis. The initial accumulation of the decarboxylase (0-9 hr) in cultures treated with "nonmitogenic" levels of concanavalin A (108 jig/ml) was similar to that observed in cultures stimulated with optimally mitogenic doses (18 ,Lg/ml). The early induction of ornithine decarboxylase (L-ornit ne carboxy-lyase, EC 4.1.1.17) was also similar under these two culture conditions. However, the second phase of adenosylmethionine decarboxylase accumulation, the induction of thymidine kinase (ATP: thymidine 5'-phosphotransferase, EC 2.7.1.21), and DNA replication were blocked at the higher concentrations of concanavalin A. The inhibition of late events by high doses of concanavalin A was reversible. Cells treated with a-methyl-Dmannopyranoside 25 hr after addition of a high dose of lectin responded with a second period of adenosylmethionine decarboxylase accumulation, induction of thymidine kinase, and progression through S phase. These results sugest that initial lymphocyte activation occurs normally at high doses of concanavalin A, but that the cells are reversibly blocked prior to induction of "late" enzymes and progression through S phase.