Control of growth of benzo(a)pyrene-transformed 3T3 cells.

114

The control of eucaryotic cell proliferation is governed largely by a series of regulatory events which occur in the Gl phase of the cell cycle. When stimulated to proliferate, quiescent (GO) 3T3 fibroblasts require transcription, rapid translation, and three growth factors for the growth state transition. We examined exponentially growing 3T3 cells to relate the requirements for Gl transit to those necessary for the transition from the GO to the S phase. Cycling cells in the Gl phase required transcription, rapid translation, and a single growth factor (insulin-like growth factor [IGF] I) to initiate DNA synthesis. IGF I acted post-transcriptionally at a late Gl step. All cells in the Gl phase entered the S phase on schedule if either insulin (hyperphysiological concentration) or IGF I (subnanomolar concentration) was provided as the sole growth factor. In medium lacking all growth factors, only cells within 2 to 3 h of the S phase were able to initiate DNA synthesis. Similarly, cells within 2 to 3 h of the S phase were less dependent on transcription and translation for entry into the S phase. Cells responded very differently to inhibited translation than to growth factor deprivation. Cells in the early and mid-Gl phases did not progress toward the S phase during transcriptional or translational inhibition, and during translational inhibition they actually regressed from the S phase. In the absence of growth factors, however, these cells continued progressing toward the S phase, but still required IGF at a terminal step before initiating DNA synthesis. We conclude that a suboptimal condition causes cells to either progress or regress in the cell cycle rather than freezing them at their initial position. By using synchronized cultures, we also show that in contrast to earlier events, this final, IGF-dependent step did not require new trnscription. This result is in conrast to findings that other growth factors induce new transcription. We examined the requirements for Gl transit by using a chemically transformed 3T3 cell line (BPA31 cells) which has lost some but not all ability to regulate its growth. Early-and mid-Gl-phase BPA31 cells required transcription and translation to initiate DNA synthesis, although they did not regress from the S phase during translational inhibition. However, these cells did not need IGF for entry into the S phase.Eucaryotic cell growth is controlled by regulatory events which occur before the onset of DNA synthesis (2, 14, 23). The presynthesis or Gi phase of the cell cycle is generally viewed as a period during which cells prepare to initiate a round of DNA replication and cell division. In addition, cells in the Gl phase assess the feasibility of these processes in regard to the extracellular environment.Much of our understanding of the nature of growth regulation derives from studies with cultured murine 3T3 fibroblasts. These established, nontumorigenic embryonic cells show a stringent degree of growth control in culture, depending on the environment into which they are placed...

Section: Methodsmentioning

confidence: 99%

Section: Downloaded Frommentioning

confidence: 99%

Post-transcriptional control of the onset of DNA synthesis by an insulin-like growth factor.

Campisi¹,

Pardee²

1984

114

“…Mouse BALB/c 3T3 cells (clone A31) and their benzo(a)pyrenetransformed derivative (BPA31) (25) were maintained in 75-cm2 tissue culture flasks (Becton Dickinson Labware, Oxnard, Calif.) in the Dulbecco modification of Eagle minimal medium (DME) supplemented with 3.7 g of sodium bicarbonate per liter, 4 mM glutamine, and 10% calf serum (Flow Laboratories, Inc., McLean, Va.). Cultures were incubated at 37°C in a water-saturated atmosphere containing 10% CO2.…”

Section: Methodsmentioning

confidence: 99%

Growth-dependent synthesis of c-myc-encoded proteins: early stimulation by serum factors in synchronized mouse 3T3 cells.

Persson¹,

Gray²,

Godeau³

1985

Synthesis of the c-myc gene product was measured during the entire cell cycle of subconfluent mouse 3T3 cells with an antibody raised against a human c-myc synthetic peptide. The antiserum recognized two mouse c-myc-encoded proteins with apparent molecular weights in sodium dodecyl sulfate-polyacrylamide gels of 62,000 and 60,000. Cell-derived p62 was compared with the mouse c-myc gene product synthesized in vitro. Immunoprecipitation, electrophoretic analyses, and peptide mapping provided evidence that p62 is encoded by the mouse c-myc gene. The rate of synthesis of the c-myc proteins was tightly coupled to the cellular growth state of nontransformed A31 3T3 cells, but not to that of their benzo(a)pyrene-transformed derivative (BPA31). Furthermore, the synthesis of the c-myc proteins was stimulated by the exposure of confluent, density-arrested A31 cells to platelet-derived growth factor or fibroblast growth factor. Tightly synchronized cell populations were obtained on the addition of serum factors to subconfluent, serum-deprived A31 cells, and c-myc expression could be monitored for more than one complete cell cycle. One hour after stimulation the steady-state level of the 2.2 kilobase c-myc transcript increased 30-fold relative to that of quiescent cells and decreased thereafter to the level observed during exponential growth. The rate of synthesis of c-myc-encoded proteins was determined by immunoprecipitation after a 2-h labeling period. After an initial sevenfold increase detectable 2 h after serum addition, the rate of synthesis remained constant throughout the rest of the cell cycle. No further changes associated with the late prereplicative period, S phase, G2, or mitosis could be demonstrated. Pulse-chase and long-term labeling experiments revealed different half-lives for the two c-myc-encoded proteins. The half-lives of the c-myc proteins, however, were independent of the cellular growth state. The sustained expression observed throughout the cell cycle suggests that the growth-related function of c-myc may be required during the GO-GI transition and in all phases of the cycle of the growing cell.

“…BPA31 cells are tumorigenic, chemically transformed A31-derived cells that enter the Go phase when deprived of serum (5,6,19). These cells have lost the IGF-I requirement for late G, progression (6) c-rais-Ha mRNA was somewhat higher in confluent cells than in serum-deprived cells.…”

Section: Characterization Of C-ras-ha Inductionmentioning

confidence: 99%

c-ras-Ha gene expression is regulated by insulin or insulinlike growth factor and by epidermal growth factor in murine fibroblasts.

Lu¹,

Levine²,

Campisi³

1989

Although much is known about the structure of ras-encoded proteins, little is known about how expression is regulated. In serum-stimulated murine fibroblasts, c-ras-Ha mRNA levels fluctuated with the growth state but not with the position in the cell cycle. Two types of growth factors regulated c-ras-Ha expression: insulin (IN) Qr insulinlike growth factor I, each apparently acting through its cognate receptor, and epidermal growth factor (EGF). In quiescent cells, IN or insulinlike growth factor I induced c-ras-Ha mRNA three-to fivefold within 4 h, but thereafter the mRNA declined. By contrast, EGF had little effect in 4 h but induced the mRNA after 4 to 6 h. When quiescent cells were given serum c-ras protooncogenes are normal cellular genes that are expressed in most cell types, both proliferating and terminally differentiated, but have the potential to play a causative role in the neoplastic transformation of certain cells (2). ras genes encode a family of membrane-associated proteins (p21 proteins) that bind and hydrolyze GTP. Structural and biochemical analyses suggest that p21 proteins function analogously to GTP-binding proteins (G proteins) that couple occupied receptors to intracellular signalling systems (20). The receptors coupled to ras are unknown, but they are presumed to deliver positive mitogenic signals.The primary mechanism by which ras protooncogenes are converted to transforming genes is a point mutation at one of three major sites in the coding sequence (2). In most cases, these point mutations diminish or abolish GTPase activity, which by analogy with other G proteins, should enhance receptor-generated signals. Indeed, when ras proteins bearing one of these point mutations are introduced into mammalian fibroblasts, the cells initiate DNA synthesis and undergo phenotypic changes characteristic of growth factorstimulated or transformed cells (15,22,36).Depending upon the cell type, the oncogenic potential of ras genes can also be manifest when the protooncogene is overexpressed (7,31,34). For example, a point mutation in an intron of the c-ras-Ha gene is apparently responsible for elevated expression of this gene in the human bladder carcinoma cell line T24/EJ; although the c-ras-Ha gene in T24/EJ cells also carries a mutation that diminishes GTPase activity, the mutation that confers overexpression is at least partially responsible for the transforming activity of the c-ras-Ha gene in these cells (10 sis when microinjected into fibroblasts that have been stimulated to proliferate by serum growth factors (26). Together, these findings suggest that the level of c-ras-Ha expression must be carefully controlled for regulated proliferation of normal cells.Despite the importance of c-ras expression for normal cell proliferation, the physiological signals that regulate these genes are largely unknown. This is in striking contrast to c-fos and c-mnc protooncogenes, which encode nuclear proteins (3) and for which the mechanisms of regulation by growth factors and intracellular mediators of growth ...