Steven M. Jones

Growth-factor-dependent mitogenesis requires two distinct phases of signalling

Prolonged and continuous exposure to growth factors is required to commit cells to the cell cycle. Here we show that the prolonged requirement for growth factor can be replaced with two short pulses of mitogen. The first pulse of growth factor moves the cell through the initial segment of the G0 to S interval. This initial pulse also makes cells responsive to a second pulse of growth factor, which engages components of the cell-cycle machinery necessary for progression into S phase. We also show that activation of MAP kinase kinase (MEK) and induction of the transcription factor c-Myc are sufficient to drive the first, but not the second, phase of signalling. Furthermore, synthetic phosphatidylinositol-3-OH kinase (PI(3)K) lipid products are sufficient to drive the second phase of signalling, but not the first. These findings suggest that there is a common signalling cascade by which mitogens drive arrested cells into the cell cycle, and that this cascade involves the temporally coordinated input of MEK, c-Myc and PI(3)K.

PDGF induces an early and a late wave of PI 3-kinase activity, and only the late wave is required for progression through G1

BACKGROUND Platelet-derived growth factor (PDGF) triggers cytoskeletal rearrangements and chemotaxis within minutes. These events are at least in part due to the activation of phosphoinositide (PI) 3-kinase; there is good temporal correlation between these events and the accumulation of 3-phosphorylated products of the kinase. Prolonged and continuous PDGF exposure results in S-phase entry many hours after the initial burst of activity. Although early signals appear responsible for the early responses, they may not fully account for later responses, such as cell-cycle progression. RESULTS We assessed when PI 3-kinase products accumulate in PDGF-stimulated cells. In addition to the previously identified early accumulation of products, we detected a second, prolonged wave of accumulation 3-7 hours after stimulation. To determine the relative contribution of each phase to PDGF-dependent DNA synthesis, we first developed an assay in which synthetic 3-phosphorylated lipids were used to rescue DNA synthesis in cells expressing a PDGF-receptor mutant. The lipids rescued DNA synthesis only when added 2-6 hours after PDGF. In addition, PI 3-kinase inhibitors failed to block PDGF-dependent DNA synthesis if added during the first wave of PI 3-kinase activity, but adding them later, in G1 phase, prevented PDGF-dependent cell-cycle progression. CONCLUSIONS PDGF induces distinct waves of PI 3-kinase activity. The second wave is required for PDGF-dependent DNA synthesis, whereas the initial wave is not. One of the ways in which cells use PI 3-kinase to mediate distinct cellular responses seems to be by regulating when its products accumulate.

Growth factor-dependent signaling and cell cycle progression

There are three central ideas contained within this review. Firstly, growth factor‐stimulated signaling is not restricted to a 30–60 min window, but occurs at a much later time as well. Secondly, the second wave of signaling overlaps temporally with the cell cycle program and may be directly responsible for engaging it. Thirdly, the G1 to S interval appears to encompass two distinct phases of the cell cycle, during which the coordinated activation of distinct sets of signaling enzymes drives cell cycle progression. Each of these concepts is likely to initiate new investigation and hence provide additional insight into the fundamental question of how growth factors drive cell proliferation.

Connecting signaling and cell cycle progression in growth factor-stimulated cells.

A widely used model system to investigate cell proliferation is stimulation of serum-arrested cells with growth factors. Recent data suggest that there are two waves of growth factor-dependent signaling events required for a proliferative response. One is an acute burst of signaling, which occurs immediately after growth factor stimulation and lasts for 30–60 min. The other occurs in a different time frame (8–12 h post stimulation), and involves activation of cyclin dependent kinases (Cdks). In addition to a general overview of growth factor-dependent signaling, we present our ‘two wave’ hypothesis for how signaling and cell cycle progression are linked.

PDGF initiates two distinct phases of protein kinase C activity that make unequal contributions to the G0 to S transition

BACKGROUND Platelet-derived growth factor (PDGF) promotes cell-cycle progression by engaging signaling enzymes such as phospholipase Cgamma (PLCgamma). When activated, PLCgamma cleaves phosphatidylinositol-4,5-bisphosphate to produce inositol-1,4, 5-trisphosphate (IP(3)) and diacylglycerol (DAG). IP(3) stimulates the release of calcium from intracellular stores, which together with DAG activate some protein kinase C (PKC) family members. In this study we focused on putative downstream effectors of PLCgamma - PKC family members. We investigated whether, and when, DAG-responsive PKCs contribute to PDGF-dependent DNA synthesis. RESULTS In HepG2 cells expressing wild-type PDGF beta receptors (betaPDGFRs), PDGF activated at least one PKC family member (PKCepsilon) at two distinct times - within 10 minutes after PDGF stimulation, and then for a longer duration between 5 and 9 hours. Blocking the early burst of PKC activity had no effect on PDGF-dependent DNA synthesis. In contrast, the DNA-synthesis response was reduced by 60-80% when the second phase of PKC activity was blocked. Similarly, DAG rescued PDGF-dependent DNA synthesis in the cells expressing a mitogenically incompetent mutant betaPDGFR, but only when DAG was added at times corresponding to the late phase of PKC activity. Our studies also indicate that the late phase of PKCepsilon activity can be induced by either phosphoinositide 3-kinase-dependent or DAG-dependent pathways in PDGF-stimulated HepG2 cells. CONCLUSIONS We conclude that PDGF activates PKCs at two distinct times and that these two intervals of PKC activity make unequal contributions to the mitogenic response. The late phase of PKC activity is required for PDGF-dependent DNA synthesis, whereas the early phase of activity is dispensable.

Influence of scheduling on two-drug combinations of alkylating agents in vivo

SummaryThe effects of schedule and sequence on the survival of EMT6 tumor cell and bone marrow (CFU-GM) obtained after treatment using combinations of cyclophosphamide (CTX) and thiotEPA or melphalan (l-PAM) were examined and analyzed by isobologram methodology. On a single-injection schedule, when CTX and thiotEPA were given simultaneously or thiotEPA was given prior to CTX, the result was slightly greater than additive tumor-cell kill. However, when CTX preceded thiotEPA by 4 h, there was less than additive cell kill. When the interval between the administration of the two drugs was 8 h, both sequences of the drugs produced greater than additive tumor-cell kill. Simultaneous administration of CTX and thiotEPA on a multiple-injection schedule resulted in sub-additive tumor-cell kill. On the multiple-injection schedule, extending the interval between injections of CTX and thiotEPA to 4 and 8 h resulted in increasing tumor-cell kill. With the 4- and 8-h intervals, no significant sequence-dependent difference in tumor-cell kill was obtained. The results of CTX andl-PAM combinations paralleled those of CTX and thiotEPA. Bone marrow (CFU-GM) survival was used as a representative normal tissue with which to compare tumor-cell survival after each treatment to obtain a measure of therapeutic effect. The trends for the ratios of bone marrow: tumor cell survival were the same for the treatment sequences of CTX with thiotEPA orl-PAM; however, greater magnitudes of differential tumor-cell kill were obtained with CTXl-PAM combinations. Using this measure, the greatest therapeutic effectiveness was seen with single-dosel-PAM or thiotEPA followed 4 h later by CTX and with CTX given as a single or as multiple doses followed 8 h later byl-PAM or thiotEPA. Such data from tumor-model systems may be useful in the development of more effective alkylating agent regimens for use in the clinic.

Influence of scheduling, dose, and volume of administration of a perfluorochemical emulsion on tumor response to radiation therapy

Studies were carried out with a new, concentrated perfluorochemical emulsion (PFCE) of the perfluorochemical F44E (48% V/V). When given at 4, 1.6, or 1 g/kg in undiluted injection volumes iv 1 hr prior to a range of single doses of radiation with inspired carbogen dose modifying factors (DMFs) based on tumor growth delay (TGD) in the Lewis lung tumor of 2.5, 1.7, and 1.5, respectively, were produced. When the PFC dose was administered in a volume of 0.2 ml, the dose modifying factors produced by 4 g/kg (0.1 ml undiluted) did not change significantly (2.6), but the dose modifying factors produced by 1.6 g/kg (0.04 ml undiluted) and by 1.0 g/kg (0.025 ml undiluted) increased significantly to 2.0 and 1.8 (p less than 0.05), respectively. Using the tumor excision assay at 24 hr post treatment in the FSaIIC fibrosarcoma, administration of 6, 4, or 2 g/kg in 0.2 ml injections plus carbogen breathing 1 hr prior to and during treatment resulted in dose modifying factors of 1.5, 1.6, and 1.3, respectively. In a fractionated radiation protocol in the Lewis lung tumor using four daily fractions, a dose of 4 g/kg of PFC on days 1 and 3 proved superior to a dose of 2 g/kg daily (dose modifying factors 2.4 vs. 1.9, p less than 0.05). When a fractionated radiation regimen of 3 Gy daily X 5 and carbogen was used, PFC doses of 0.5, 1, 2, and 4 g/kg administered undiluted produced increasing tumor growth delays with increasing dose of PFCE and increasing frequency of administration. In addition, dilutions to 0.2 ml proved significantly more effective. In a 2-week fractionated radiation protocol using 2, 3, or 4 Gy daily X 5 weekly, PFCE given in 0.2 ml volume plus carbogen breathing daily at 4, 1.6, or 1 g/kg produced dose modifying factors of 2.0, 1.9, and 1.6, respectively. Finally, when used in a day 1, 3, and 5 radiation regimen for 3 weeks at 2, 3, or 4 Gy/fraction, 4 g/kg of PFCE given in a volume of 0.2 ml plus carbogen breathing produced a superior dose modifying factor (1.6) as compared with 1.6 or 1.0 g/kg (dose modifying factors 1.4 and 1.3, respectively). These results indicate that PFCE plus carbogen breathing effectively enhances the antitumor effects of both single dose and fractionated radiation.(ABSTRACT TRUNCATED AT 400 WORDS)