Comparative pharmacokinetics of RAD001 (everolimus) in normal and tumor-bearing rodents.

PURPOSE: Comparative pharmacokinetic (PK) analysis of the mTOR inhibitor RAD001 (everolimus) in rats and mice. METHODS: Blood cell partitioning, plasma protein binding and PK parameters of RAD001 in blood and tissues (including brain) of both mice and rats were determined. PK modeling predicted plasma/blood and tumor levels from a variety of regimens and these were compared with the known human PK profile. DCE-MRI was used to compare tumor vascularity between mice and rats. Estimation of IC50 values in vitro and ED50 values in vivo were used to provide an indication of anti-tumor activity. RESULTS: The PK properties of RAD001 differed between mice and rats, including erythrocyte partitioning, plasma protein binding, plasma/blood t(1/2), oral bioavailability, volume of distribution, tissue/tumor penetration and elimination. Modeling of tumor and blood/plasma PK suggested that in mice, multiple daily administrations result in a 2-fold increase in tumor levels of RAD001 at steady state, whereas in rats, a 7.9-fold increase would occur. Weekly high-dose regimens were predicted not to facilitate tumor accumulation in either species. Total tumor levels of RAD001 were four- to eight-fold greater in rats than in mice. Rat tumors had a >2-fold greater plasma content and permeability compared to mouse tumors, which could contribute to differences in tumor drug uptake. Maximal antitumor effects (T/C of 0.04-0.35) were observed in both species after daily administration with similar C(max) and AUC values of unbound (free) RAD001. These free levels of RAD001 are exceeded in serum from cancer patients receiving clinically beneficial daily regimens. In rodents, brain penetration of RAD001 was poor, but was dose-dependent and showed over-proportional uptake in rats with a longer t(1/2) compared to the systemic circulation. CONCLUSIONS: The PK of RAD001 differed between mice and rats, with rats having a PK profile closer to that of humans. High intermittent doses of RAD001 may be more appropriate for treatment of brain tumors.

Impaired vasoreactivity in bodybuilders using androgenic anabolic steroids.

BACKGROUND: Anabolic androgenic steroids are used by some bodybuilders to enhance performance. While the cardiovascular implications of supraphysiological androgen levels requires further clarification, use is associated with sudden death, left ventricular hypertrophy, thrombo-embolism and cerebro-vascular events. MATERIALS AND METHODS: To further understand the effect of androgenic anabolic steroid abuse on vascular function, this study assessed vascular stiffness (pulse-wave analysis) and cardiovascular risk factors in 28 male, bodybuilding subjects, of whom ten were actively receiving anabolic agents (group A; 26.4 +/- 7.2 years) and eight had undergone a 3-month "wash-out" period (group B; 32.1 +/- 7.1 years). The remaining ten bodybuilding subjects (group C; 24.4 +/- 4.4 years) denied any past use of anabolic steroids or other performance enhancing drugs. Comparisons were made with ten sedentary male controls (group D, 29.3 +/- 4.7 years). RESULTS: Endothelial independent dilatation in response to glycerol trinitrate was significantly impaired in the group currently using anabolic steroids (group A) compared with the other three groups [A (5.63 +/- 3.24%) versus; B (11.10 +/- 4.91%), C (17.88 +/- 9.2%) and D (14.46 +/- 3.9%), P < 0.0005, respectively], whereas no significant differences in endothelial-dependent dilatation were detected between the groups [A (5.0 +/- 3.0%), B (7.4 +/- 3.4%), C (9.6 +/- 4.5%) and D (8.2 +/- 3.3%), P < 0.059, respectively]. CONCLUSIONS: Previous studies described a decline in vascular reactivity occurring in bodybuilding subjects which is independent of anabolic steroid use and may result from smooth muscle hypertrophy with increased vascular stiffness. This study revealed impaired vascular reactivity associated with anabolic agents and that improvement in vascular function may occur following their discontinuation.

Endothelial function and coagulant factors in growth hormone-treated hypopituitary adults receiving desmopressin.

Although GH deficiency may underlie the increased cardiovascular risk in adult hypopituitarism, other coexisting hormonal deficiencies and/or unphysiological hormone replacement may contribute. L-Deamino-8-D-arginine (DDAVP), when administered parenterally, potentiates hemostasis by increasing plasma procoagulant factors. We investigated whether chronic intranasal DDAVP therapy influences clotting factors (plasma fibrinogen, factor VIII, and von Willebrand factor antigen) and endothelial function (flow-mediated dilation of the brachial artery) in 30 GH-treated hypopituitary subjects, including both DDAVP-treated subjects (group A) (mean age, 46 +/- 11 yr) and vasopressin-sufficient subjects (group B) (mean age, 47 +/- 16 yr). Fifteen healthy controls (group C) (mean age, 48 +/- 12 yr) were also studied. All hypopituitary patients were receiving stable GH replacement (median duration, 19 months). Comparing the three groups, concentrations of fibrinogen (mean +/- SD) (A, 3.3 +/- 1.0 g/liter vs. B, 3.5 +/- 0.9 vs. C, 2.6 +/- 0.8, P < 0.05), factor VIII (A, 130% +/- 30% vs. B, 128% +/- 30% vs. C, 104% +/- 35%, P < 0.05) and von Willebrand factor antigen (A, 124% +/- 35% vs. B, 134% +/- 45% vs. C, 93% +/- 36%, P < 0.05) were higher in hypopituitary subjects, compared with controls. However, there were no differences in clotting factors between groups A and B. Flow-mediated dilation did not differ significantly between the two hypopituitary groups (A, 5.9% +/- 2.0% vs. B, 4.7% +/- 1.6%) and was similar to that in the control group (C, 5.7% +/- 2.1%). In conclusion, although endothelium-dependent vasodilation is intact in GH-treated hypopituitary adults, elevated concentrations of hemostatic markers suggest the persistence of a prothrombotic tendency and endothelial dysfunction. Intranasal DDAVP does not appear to influence this proatherogenic profile in hypopituitary adults with vasopressin deficiency.

Modulation of p27/Cdk2 complex formation through 4D5-mediated inhibition of HER2 receptor signaling.

The molecular mechanisms mediating the anti-proliferative effects of the murine anti-HER2 monoclonal antibody (4D5) were investigated in HER2-overexpressing human carcinoma cell lines. Treatment with 4D5 resulted in a dramatic accumulation of BT-474 breast carcinoma cells in G1; concomitant with reduced expression of proteins involved in sequestration of the cyclin E/Cdk2 inhibitor protein p27, increased association of p27 with Cdk2 complexes and Cdk2 inactivation. No equivalent effects were observed in BT-474 cells treated with a control, non-inhibitory HER2 monoclonal antibody (FRP5) or in a HER2-overexpressing cell line insensitive to 4D5 treatment (MKN7 gastric carcinoma cells), confirming the relationship between these molecular changes and 4D5-mediated inhibition of proliferation. Increased p27 expression was also observed in 4D5-treated BT-474 cells; however an antisense approach demonstrated that this increase was not required for Cdk2 inactivation or establishment of the G1 block. These data suggest that 4D5 interferes with HER2 receptor signaling, resulting in downregulation of proteins involved in p27 sequestration. This causes release of p27, allowing binding and inhibition of cyclin E/Cdk2 complexes and inhibition of G1/S progression. This model was confirmed using a second 4D5-sensitive. HER2-overexpressing breast tumor line (SKBR3), and suggests that the dependency of a given tumor cell on elevated HER2-receptor signaling for the maintenance of p27 sequestration proteins may determine the clinical response to treatment with the humanized anti-HER2 monoclonal antibody Herceptin (trastuzumab).

The role of overexpressed HER2 in transformation.

The HER family of receptors has an important role in the network of cell signals controlling cell growth and differentiation. Although the activity of the HER receptor is strictly controlled in normal cells, HER2 receptor overexpression plays a pivotal role in transformation and tumorigenesis. HER2 gene amplification and/or overexpression of the receptor has been detected in subsets of a wide range of human cancers including breast cancer, and is an indicator of poor prognosis. It is proposed that overexpressed HER2 in combination with HER3 causes high activity of cell-signaling networks, thereby resulting in tumor cell proliferation. Thus, the HER2 receptor is an attractive target for new anti-cancer treatments. Monoclonal antibodies directed against the receptor are the most promising of these, and the humanized anti-HER2 monoclonal antibody trastuzumab (Herceptin) has shown significant clinical efficacy in clinical trials. The anti-tumor mechanisms of anti-HER2 monoclonal antibodies are not completely understood. However, some tumor types are not sensitive to trastuzumab, suggesting that the response of a tumor to trastuzumab may not only be dependent on overexpressed HER2, but may also be influenced by other members of the HER receptor family expressed in the tumor cell.

Myc and mammary cancer: Myc is a downstream effector of the ErbB2 receptor tyrosine kinase.

The proto-oncogene c-myc encodes a transcription factor which plays a major role in the regulation of normal cellular proliferation and is aberrantly expressed in many breast tumors. In a normal cell Myc expression levels are tightly regulated being subject to many layers of control. Errantly expressed Myc collaborates with other oncogenes to promote transformation. In this review we will focus on the association between abnormal Myc expression and mammary cancer. In particular, we will discuss the role of Myc as a downstream effector of the ErbB2 receptor tyrosine kinase which is overexpressed and constitutively activate in many mammary tumors. The cooperation between Myc and ErbB2 in transformation will be discussed in relation to clinical studies on Myc in human cancer and with consideration of transgenic models of Myc-induced mammary cancer. Data from our laboratory will be presented showing that deregulated ErbB2 activity strongly stimulates cytoplasmic signaling pathways which in turn impinge on Myc at multiple levels causing its deregulated expression.

Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells.

The ErbB2 receptor tyrosine kinase is overexpressed in a variety of human tumours. In order to understand the mechanism by which ErbB2 mediates tumour proliferation we have functionally inactivated the receptor using an intracellularly expressed, ER-targeted single-chain antibody (scFV-5R). Inducible expression of scFv-5R in the ErbB2-overexpressing SKBr3 breast tumour cell line leads to loss of plasma membrane localized ErbB2. Simultaneously, the activity of ErbB3, MAP kinase and PKB/Akt decreased dramatically, suggesting that active ErbB2/ErbB3 dimers are necessary for sustained activity of these kinases. Loss of functional ErbB2 caused the SKBr3 tumour cells to accumulate in the G1 phase of the cell cycle. This was a result of reduction in CDK2 activity, which was mediated by a re-distribution of p27Kip1 from sequestering complexes to cyclin E/CDK2 complexes. The level of c-Myc and D-cyclins, proteins involved in p27KiP1 sequestration, decreased in the absence of functional ErbB2. Ectopic expression of c-Myc led to an increase in D cyclin levels, CDK2 activity and resulted in a partial G1 rescue. We propose that c-Myc is a primary effector of ErbB2-mediated oncogenicity and functions to prevent normal p27Kip1 control of cyclinE/CDK2.

ErbB2 potentiates breast tumor proliferation through modulation of p27(Kip1)-Cdk2 complex formation: receptor overexpression does not determine growth dependency.

Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G(1) arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27(Kip1), and inactivation of cyclin-Cdk2 complexes. Time courses demonstrated that 4D5 treatment redirects p27(Kip1) onto Cdk2 complexes, an event preceding increased p27(Kip1) expression; this correlates with the downregulation of c-Myc and D-type cyclins (proteins involved in p27(Kip1) sequestration) and the loss of p27(Kip1) from Cdk4 complexes. Similar events were observed in ErbB2-overexpressing SKBR3 cells, which exhibited reduced proliferation in response to 4D5 treatment. Here, p27(Kip1) redistribution resulted in partial Cdk2 inactivation, consistent with a G1 accumulation. Moreover, p27(Kip1) protein levels remained constant. Antisense-mediated inhibition of p27(Kip1) expression in 4D5-treated BT474 cells further demonstrated that in the absence of p27(Kip1) accumulation, p27(Kip1) redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G(1) block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27(Kip1) sequestration proteins, thus deregulating the G(1)/S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.

NDF/heregulin-induced cell cycle changes and apoptosis in breast tumour cells: role of PI3 kinase and p38 MAP kinase pathways.

Neu differentiation factor (NDF)/heregulin activates ErbB2 via heterodimerization with the NDF receptors ErbB3 and ErbB4. Cells which express normal levels of these receptors are often growth stimulated by NDF, whereas SKBR3, and other ErbB2-overexpressing breast tumour cells are growth inhibited. We demonstrate here that in SKBR3 cells, NDF induces G1 progression but also causes a G2 delay from day 1 and apoptosis from days 2-3. G1 progression was associated with ErbB2 transactivation of ErbB3 and subsequent stimulation of the phosphatidylinositol 3-kinase (PI3K) pathway whereas apoptosis was dependent on p38 MAPK. Inhibition of ERK1/ERK2 had no effect on cell cycle progression or apoptosis. Activation of ErbB3 and PI3K was also seen with betacellulin (BTC) but not epidermal growth factor (EGF) and correlated with the growth effects of these ligands. All three ligands induced short-term activation of p38 MAPK in a c-Src-dependent manner. However, only NDF caused a second, c-Src-independent increase in p38 MAPK activity which was required for apoptosis.

On the regulation and function of human polo-like kinase 1 (PLK1): effects of overexpression on cell cycle progression.

The human protein kinase Plk1, a member of the polo-like kinase family, is known to function at mitosis. Here we show that the relative specific activity of Plk1 increases in mitosis, that Plk1 is specifically phosphorylated during mitosis, and that phosphatase treatment reduces mitotic Plk1 activity to interphase levels. To identify domains involved in the regulation of Plk1 activity, deletion mutants of Plk1 were constructed and their activities examined. Deletion of the extreme C-terminus of Plk1 substantially increased kinase activity, indicating that the C-terminus harbors an inhibitory domain. Finally, the consequences of over-production of wild-type and mutant Plk1 protein were analyzed, using transient transfection assays. Cells overexpressing Plk1 protein were able to enter mitosis and establish an apparently normal bipolar spindle. In contrast, progression through mitosis was transiently delayed, and cytokinesis appeared to be disturbed, as reflected by a significant increase in large cells with multiple, often fragmented nuclei. These results are relevant to recently proposed roles for Plks during both entry into and exit from mitosis.

Cell-cycle control: POLO-like kinases join the outer circle.

Named after the polo gene of Drosophila, POLO-like kinases (PLKs) constitute a novel, evolutionarily conserved family of essential cell-cycle regulators. As emphasized in this review, recent studies identify important roles for vertebrate PLKs at the onset of mitosis: Plx1, a Xenopus PLK, has been implicated in the activation of Cdc25 phosphatase (and hence the activation of Cdc2), while human Plk1 is required for the proper maturation of the poles of mitotic spindles. These studies suggest a major role for Plk1/Plx1 in coordinating spindle assembly with the activation of Cdc2-cyclin complexes, and they establish a direct link between PLKs and the core cell-cycle-regulatory machinery. Genetic and biochemical studies in yeasts and Drosophila point to additional roles for PLKs at later stages of mitosis. Finally, mammals express multiple PLKs, suggesting that different family members might function at distinct cell-cycle transitions, reminiscent of cyclin-dependent kinases.

Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes.

Mammalian polo-like kinase 1 (Plk1) is structurally related to the polo gene product of Drosophila melanogaster, Cdc5p of Saccharomyces cerevisiae, and plo1+ of Schizosaccharomyces pombe, a newly emerging family of serine-threonine kinases implicated in cell cycle regulation. Based on data obtained for its putative homologues in invertebrates and yeasts, human Plk1 is suspected to regulate some fundamental aspect(s) of mitosis, but no direct experimental evidence in support of this hypothesis has previously been reported. In this study, we have used a cell duplication, microinjection assay to investigate the in vivo function of Plk1 in both immortalized (HeLa) and nonimmortalized (Hs68) human cells. Injection of anti-Plk1 antibodies (Plk1+) at various stages of the cell cycle had no effect on the kinetics of DNA replication but severely impaired the ability of cells to divide. Analysis of Plk1(+)-injected, mitotically arrested HeLa cells by fluorescence microscopy revealed abnormal distributions of condensed chromatin and monoastral microtubule arrays that were nucleated from duplicated but unseparated centrosomes. Most strikingly, centrosomes in Plk1(+)-injected cells were drastically reduced in size, and the accumulation of both gamma-tubulin and MPM-2 immunoreactivity was impaired. These data indicate that Plk1 activity is necessary for the functional maturation of centrosomes in late G2/early prophase and, consequently, for the establishment of a bipolar spindle. Additional roles for Plk1 at later stages of mitosis are not excluded, although injection of Plk1+ after the completion of spindle formation did not interfere with cytokinesis. Injection of Plk1+ into nonimmortalized Hs68 cells produced qualitatively similar phenotypes, but the vast majority of the injected Hs68 cells arrested as single, mononucleated cells in G2. This latter observation hints at the existence, in nonimmortalized cells, of a centrosome-maturation checkpoint sensitive to the impairment of Plk1 function.

Dynamic changes in nuclear architecture during mitosis: on the role of protein phosphorylation in spindle assembly and chromosome segregation.

During mitosis, the vertebrate cell nucleus undergoes profound changes in architecture. At the onset of mitosis, the nuclear envelope breaks down, the nuclear lamina is depolymerized, and interphase chromatin is condensed to chromosomes. Concomitantly, cytoplasmic microtubules are reorganized into a mitotic spindle apparatus, a highly dynamic structure required for the segregation of sister chromatids. Many of the above events are controlled by reversible phosphorylation. Hence, our laboratory is interested in characterizing the kinases involved in promoting progression through mitosis and in identifying their relevant substrates. Prominent among the kinases responsible for regulating entry into mitosis is the Cdc2 kinase, the first member of the cyclin dependent kinase (Cdk) family. Recently, we found that Cdc2 phosphorylates HsEg5, a human kinesin-related motor protein associated with centrosomes and the spindle apparatus. Our results indicate that phosphorylation regulates the association of HsEg5 with the mitotic spindle and that the function of this plus-end directed motor is essential for centrosome separation and bipolar spindle formation. Another kinase implicated in regulating progression through mitosis is Plk1 (polo-like kinase 1), the human homologue of the Drosophila gene product "polo." By antibody microinjection we have found that Plk1 is required for the functional maturation of centrosomes and hence for entry into mitosis. Furthermore, we found that microinjected anti-Plk1 antibodies caused a more severe block to cell cycle progression in diploid fibroblasts than in immortalized tumor cells. This observation hints at the existence of a checkpoint linking Cdc2 activation to the presence of functional centrosomes.

The family of polo-like kinases.

Here we discuss members of a new family of serine/threonine protein kinases with a likely role in cell cycle control. These kinases are referred to as polo-like kinases, after the prototypic founding member of the family, the polo gene product of Drosophila melanogaster. The polo kinase was originally identified in mutants that display abnormal mitotic spindle organization. Subsequently, potential homologues of Drosophila polo have been identified in yeasts (Cdc5p in Saccharomyces cerevisiae; plo1+ in Schizosaccharmoyces pombe) and in mammals (polo-like kinase 1; Plk1). Genetic and biochemical studies suggest that polo, Cdc5p and plo1+ may be required for mitotic spindle organization and, possibly, for cytokinesis. Likewise, the patterns of expression, activity and subcellular localization of Plk1 strongly suggest that this mammalian kinase functions also during mitosis, possibly in spindle assembly and function. In addition to Plk1, however, more distantly related members of the polo-like kinase family have been identified in mammalian cells, and the available data are consistent with the idea that some of these may act earlier in the cell cycle, possibly during G1. If this hypothesis is correct, different members of the polo-like kinase family would act at several points during the cell cycle, reminiscent of the behaviour of Cdk/cyclin complexes.

Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo.

We have isolated a human homolog of Xenopus Eg5, a kinesin-related motor protein implicated in the assembly and dynamics of the mitotic spindle. We report that microinjection of antibodies against human Eg5 (HsEg5) blocks centrosome migration and causes HeLa cells to arrest in mitosis with monoastral microtubule arrays. Furthermore, an evolutionarily conserved cdc2 phosphorylation site (Thr-927) in HsEg5 is phosphorylated specifically during mitosis in HeLa cells and by p34cdc2/cyclin B in vitro. Mutation of Thr-927 to nonphosphorylatable residues prevents HsEg5 from binding to centrosomes, indicating that phosphorylation controls the association of this motor with the spindle apparatus. These results indicate that HsEg5 is required for establishing a bipolar spindle and that p34cdc2 protein kinase directly regulates its localization.

p70s6k function is essential for G1 progression.

An essential step in the pathway by which growth factors trigger cellular proliferation is the induction of high levels of protein synthesis. This appears in part to be controlled by multiple phosphorylation of the ribosomal protein S6 (refs 4, 5). The main kinase responsible, p70s6k (refs 6-8), is activated through the phosphorylation of four sites clustered in a putative autoinhibitory domain, which is mediated by a signalling pathway distinct from those used by other well characterized mitogen-activated serine/threonine kinases (such as p42/p44mapk or p90rsk; refs 10, 11). Here we investigate the role of p70s6k in the mitogenic response. Microinjection of quiescent rat embryo fibroblasts with any of three distinct polyclonal antibodies to p70s6k abolishes serum-induced entry into S phase of the cell cycle. This effect is preceded by almost complete abrogation of the activation of protein synthesis and the expression of an essential immediate early gene product, c-fos. The inhibitory effect on DNA synthesis is also elicited by microinjection of the antibodies late in G1 phase, consistent with the finding that p70s6k activity remains high throughout G1.

Identification and early activation of a Xenopus laevis p70s6k following progesterone-induced meiotic maturation.

Employing oligonucleotide primers derived from the DNA sequence of rat p70s6k a homologous sequence was shown by polymerase chain reaction (PCR) to be present as a maternal transcript in stage IV-VI Xenopus laevis oocytes. The sequence covered 665 bp of p70s6k and was 97% identical at the amino acid level. When used to probe a Northern blot of the poly(A)+ mRNA from stage VI oocytes, this sequence recognized four transcripts of 1.9, 2.5, 3.2 and 5.2 kb. Specific rat p70s6k antibodies immunoprecipitated active S6 kinase from stage VI oocytes but not unfertilized eggs. The basal level of activity was 3- to 5-fold higher in primed versus non-primed oocytes indicating that p70s6k activation was an early event in maturation. Consistent with this observation, progesterone induced a 10-fold activation of the kinase in non-primed oocytes within 1 h post-induction at a time critical for early activation of protein synthesis. A much smaller, variable peak of activation was observed at 85% germinal vesicle breakdown (GVBD), which was dependent on the rate of maturation. Two members of the pp90rsk family, thought to be the sole S6 kinases present in X.laevis oocytes, exhibited distinct kinetics of activation. Finally, the S6 kinase activity present 1 h post-progesterone stimulation was purified and shown to have a Mr of 70K.

Inhibition or down-regulation of protein kinase C attenuates late phase p70s6k activation induced by epidermal growth factor but not by platelet-derived growth factor or insulin.

The late phase of the time-dependent epidermal growth factor (EGF)-induced biphasic activation of the p70s6k is selectively attenuated by the specific PKC inhibitor, CGP 41,251, a staurosporine derivative. At a 40-fold lower concentration than CGP 41,251, staurosporine inhibits both phases of S6 kinase activation to the same extent, whereas the inactive staurosporine derivative CGP 42,700 shows no effect on either phase. Platelet-derived growth factor (PDGF) and insulin also induce biphasic S6 kinase activation, but in neither case is either phase of activation affected by the presence of CGP 41,251. This finding was unexpected in the case of PDGF, which is a potent activator of PKC and whose receptor directly interacts with phospholipase C gamma 1. However, similar results were obtained following down-regulation of PKC by prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Therefore, even though EGF and PDGF induce PKC activation, PDGF, unlike EGF, does not appear to use this signaling pathway for late phase p70s6k activation.