Redox-mediated bypass of restriction point via skipping of G1pm

Theoretical Biology and Medical Modelling Redox-mediated bypass of restriction point via skipping of G1pm Arnold Hoffman 2 James J Greene 1 Lee M Spetner 2 Michael Burke 0 2 0 Tel Aviv Sourasky Medical Center , Tel Aviv , Israel 1 Catholic University of America , Washington, DC , USA 2 Redoxia , Jerusalem , Israel Background: It is well known that cancer cells bypass the restriction point, R, and undergo uncontrolled cell proliferation. Hypothesis and evidence: We suggest here that fibrosarcoma cells enter G1ps directly from M, skipping G1pm, hence bypassing R, in response to redox modulation. Evidence is presented from the published literature that demonstrate a shortening of the cycle period of transformed fibroblasts (SV-3T3) compared to the nontransformed 3T3 fibroblasts, corresponding to the duration of G1pm in the 3T3 fibroblasts. Evidence is also presented that demonstrate that redox modulation can induce the CUA-4 fibroblasts to bypass R, resulting in a cycle period closely corresponding to the cycle period of fibrosarcoma cells (HT1080). Conclusion: The evidence supports our hypothesis that a low internal redox potential can cause fibrosarcoma cells to skip the G1pm phase of the cell cycle. - Background The normal cell cycle consists of four main phases; G1, S, G2 and M. G1 is further subdivided into two parts, G1pm and G1ps [1]. In G1pm, a series of mitogenic events prepares the cell to enter G1ps and to continue to S and M [1,2]. At the end of G1pm, there is a restriction point, R, which monitors the cell and checks its qualifications for entry into G1ps. If the accumulation of mitogenic events is inadequate, or if the cell is confluent with neighboring cells fully around its perimeter, the cell cannot pass from G1pm through R into G1ps and proliferate. Instead, the cell leaves the cell cycle and enters G0, the quiescent phase [1-5]. Cancer cells, on the other hand, bypass R with consequent uncontrolled proliferation [2]. Zetterberg and Larsson demonstrate that the transformed 3T3 cells, SV-3T3, behave in a similar way [3,4]. Furthermore, they demonstrate that these transformed cells do not enter G0. They conclude from this that tumor cells do not enter G0 [4]. Zetterberg and Larsson [1] have measured the duration of both G1pm and the complete cell cycle. Larsson and Zetterberg [3] have determined the cycle period of SV-3T3 cells. From the data in [1] and [3], we calculate that the difference between the cycle periods of the 3T3 and SV-3T3 cells is 23%; i.e. the cycle period of SV-3T3 cells is 23% shorter than that of 3T3 cells and matches the duration of G1pm. We hypothesize here that the 23% decrease in cycle period of SV-3T3 is observed because these cells skip G1pm and enter G1ps directly from the exit from M. In skipping G1pm the SV-3T3 cells bypass R. This hypothesis is supported by the following: (1) it readily accounts for the qualitative RFieglautrioen1ship between Rb brake and other aspects of cell cycle Relationship between Rb brake and other aspects of cell cycle. The Rb protein acts as a brake on several of the phases of the cell cycle, dependent upon its state of phosphorylation. In the hyperphosphorylated state, the Rb brake is inactive, permitting the transcription factors to become activated and cellular proliferation to proceed. During this period the ratio [GSSG]/[GSH] is low and E falls below . The cell passes through the restriction point R to the later stage of G1, termed G1ps, on to S, from which it passes through G2 to the early M phase. After mid-M, the Rb protein becomes hypophosphylated and the brake is active. The transcription factors are inactivated and cell proliferation is stopped. During this period the ratio [GSSG]/[GSH] is high and E rises above . The cell passes through M to the early stage of G1, termed G1pm, from which it may either return to the cell cycle via R or it passes into a resting stage, G0. In cancer, a portion of the cycle can be short-circuited, via the M to G1ps bypass. R = site of restriction point. Arrow with interrupted line represents short-circuit in cancer. = -207 11 mV. differences between non-transformed and transformed cells as noted above; and (2) it accounts for the quantitative difference between the non-transformed and transformed cell-cycle periods. The relationship between Rb brake and other aspects of cell cycle is depicted in figure 1. The mechanism we suggest for the cancer cell skipping G1pm follows from our model of redox modulation of cellular proliferation [6]. Beyond the restriction point, R, the cell is committed to duplicating its DNA and proceeding to mitosis. For a cell to pass R, special proliferation-promoting proteins must be phosphorylated to promote the activation of the genes necessary for the cell to traverse R, enter G1ps, and proliferate. These include the retinoblastoma protein (pRb) [2,5], regulatory enzymes such casein kinase [7], and transcription factors such as jun [7] and NF-B [8]. When the intracellular redox potential, E, is high, these proteins are dephosphorylated; when E is low they are phosphorylated [7-10]. An example of a critical phosphorylation-dependent pathway regulating passage through G1pm is the cyclin D-cdk4 complex. This complex phosphorylates pRb, thereby deactivating its repressor activity and allowing for transcription of S-phase genes. For this reason, the hypothesis is limited to transformed and malignant cells in which pRb is functional. According to the redox model, the dephosphorylation of pRb can occur only if the intracellular redox potential, E, is above a threshold value, , which we have estimated to be between -218 and 196 mV [6]. The cell normally sets E below when the activating proteins are to be phosphorylated, and sets E above when they are to be dephosphorylated [6] (see figure 1). During normal proliferation, when the cell is in M, a phosphatase dephosphorylates pRb [5], and the transcription factors no longer become available for activating the proliferation-promoting genes. The cell then exits M and enters G1pm and again begins to accumulate mitogenic events necessary for the cell once more to pass R and enter Doubling times were determined from cell growth curves of duplicate cultures. Cell counts were made every 24 h over a period of 96 h and the standard error in the counts was <2.5% for all curves. Normal fibroblast cultures were between passage levels of 9 to 20 passages. HT1080 passage levels were in excess of 100 passages. Although multiple, often overlapping, pathways impinge on cell-cycle regulatory points, pRb is one of the key downstream elements known to play a critical regulatory role [2,5]. Since the proper functioning of the unmutated pRb is dependent on cycling between its phosphorylated and unphosphorylated states, the redox state may contribute to altering the cell cycle by affecting pRb directly or at an upstream point. According to this redox model, if E were to be below for the du (...truncated)