Sindbis virus replication, is insensitive to rapamycin and torin1, and suppresses Akt/mTOR pathway late during infection in HEK cells

Genetically engineered Sindbis viruses (SIN) are excellent oncolytic agents in preclinical models. Several human cancers have aberrant Akt signaling, and kinase inhibitors including rapamycin are currently tested in combination therapies with oncolytic viruses. Therefore, it was of interest to delineate possible cross-regulation between SIN replication and PI3K/Akt/mTOR signaling. Here, using HEK293T cells as host, we report the following key findings: (a) robust SIN replication occurs in the presence of mTOR specific inhibitors, rapamycin and torin1 or Ly294002 – a PI3K inhibitor, suggesting a lack of requirement for PI3K/Akt/mTOR signaling; (b) suppression of phosphorylation of Akt, mTOR and its effectors S6, and 4E-BP1 occurs late during SIN infection: a viral function that may be beneficial in counteracting cellular drug resistance to kinase inhibitors; (c) Ly294002 and SIN act additively to suppress PI3K/Akt/mTOR pathway with little effect on virus release; and (d) SIN replication induces host translational shut off, phosphorylation of eIF2α and apoptosis. This first report on the potent inhibition of Akt/mTOR signaling by SIN replication, bolsters further studies on the development and evaluation of engineered SIN genotypes in vitro and in vivo for unique cytolytic functions.     

M-T5, the ankyrin repeat, host range protein of myxoma virus, activates Akt and can be functionally replaced by cellular PIKE-A

The myxoma virus (MV) ankyrin repeat, host range factor M-T5 has the ability to bind and activate cellular Akt, leading to permissive MV replication in a variety of diverse human cancer cell lines (G. Wang, J. W. Barrett, M. Stanford, S. J. Werden, J. B. Johnston, X. Gao, M. Sun, J. Q. Cheng, and G. McFadden, Proc. Natl. Acad. Sci. USA 103:4640-4645, 2006). The susceptibility of permissive human cancer cells to MV infection is directly correlated with the basal or induced levels of phosphorylated Akt. When M-T5 is deleted from MV, the knockout virus, vMyxT5KO, can no longer productively infect a subset of human cancer cells (designated type II) that exhibit little or no endogenous phosphorylated Akt. In searching for a host counterpart of M-T5, we noted sequence similarity of M-T5 to a recently identified ankyrin repeat cellular binding protein of Akt called PIKE-A. PIKE-A binds and activates the kinase activity of Akt in a GTP-dependent manner and promotes the invasiveness of human cancer cell lines. Here, we demonstrate that transfected PIKE-A is able to rescue the ability of vMyxT5KO to productively infect type II human cancer cells that were previously resistant to infection. Also, cancer cells that were completely nonpermissive for both wild-type and vMyxT5KO infection (called type III) were rendered fully permissive following ectopic expression of PIKE-A. We conclude that the MV M-T5 host range protein is functionally interchangeable with the host PIKE-A protein and that the activation of host Akt by either M-T5 or PIKE-A is critical for the permissiveness of human cancer cells for MV.     

The role of cell signaling in poxvirus tropism: the case of the M-T5 host range protein of myxoma virus

Poxviruses demonstrate strict species specificity in vivo that range from narrow to broad, however the fundamental factors that mediate the basis of poxvirus tropism remain poorly understood. It is generally believed that most, if not all, poxviruses can efficiently bind and enter a wide range of mammalian cells and all of the known host anti-viral pathways that block viral replication in nonpremissive cells operate downstream of virus entry. A productive poxvirus infection is heavily dependent upon the production of a vast array of host modulatory products that specifically target and manipulate both extracellular immune response pathways of the host, as well as intracellular signal transduction pathways of the individually infected cells. The unique pathogenesis and host tropism of specific poxviruses can be attributed to the broad diversity of host modulatory proteins they express. Myxoma virus (MV) is a rabbit-specific poxviruses that encodes multiple host range factors, including an ankyrin-repeat protein M-T5, which functions to regulate tropism of MV for rabbit lymphocytes and some human cancer cells. At the molecular level, M-T5 binds and alters at least two distinct cellular proteins: Akt and cullin-1. The direct interaction between M-T5 and Akt was shown to be a key restriction determinant for MV tropism in a spectrum of human cancer cells making MV an excellent oncolytic candidate. Thus, the intricate relationship between viral encoded proteins and components of the host cell signaling networks can have profound impact on poxvirus tropism. The lessons we continue to learn from poxvirus host range factors like M-T5 will provide further insights into the factors that regulate poxvirus tropism and the mechanisms by which poxviruses micromanipulate the signaling pathways of the infected cell.     

PI3K/Akt/mTOR信号通路及临床相关肿瘤抑制剂

哺乳动物雷帕霉素靶（mTOR）和蛋白激酶B（Akt/PKB）与肿瘤发生的密切关系已被广泛地认可.mTOR是一种丝/苏氨酸激酶,可以通过影响mRNA转录、代谢、自噬等方式调控细胞的生长.它既是PI3K的效应分子,也可以是PI3K的反馈调控因子.mTORC1 和mTORC2是mTOR的两种不同复合物. 对雷帕霉素敏感的mTORC1受到营养、生长因子、能量和应激4种因素的影响.生长因子通过PI3K/Akt信号通路调控mTORC1是最具特征性调节路径.而mTORC2最为人熟知的是作为Akt473磷酸化位点的上游激酶. 同样,Akt/PKB在细胞增殖分化、迁移生长过程中发挥着重要作用. 随着Thr308和Ser473两个位点激活,Akt/PKB也得以全面活化.因此,mTORC2-Akt-mTORC1的信号通路在肿瘤形成和生长中是可以存在的.目前临床肿瘤治疗中,PI3K/Akt/mTOR是重要的靶向治疗信号通路.然而,仅抑制mTORC1活性,不是所有的肿瘤都能得到预期控制.雷帕霉素虽然能抑制mTORC1,但也能反馈性地增加PI3K信号活跃度,从而影响治疗预后.近来发现的第二代抑制剂可以同时抑制mTORC1/2和PI3K活性,这种抑制剂被认为在肿瘤治疗上颇具前景.本综述着重阐述了PI3K/Akt/mTOR信号通路的传导、各因子之间的相互调控以及相关抑制剂的发展.     

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) enhances the efficacy of rapamycin in human cancer cells

mTOR – the mammalian/mechanistic target of rapamycin – has been implicated as a key signaling node for promoting survival of cancer cells. However, clinical trials that have targeted mTOR with rapamycin or rapamycin analogs have had minimal impact. In spite of the high specificity of rapamycin for mTOR, the doses needed to suppress key mTOR substrates have proved toxic. We report here that rapamycin when combined with AICAR – a compound that activates AMP-activated protein kinase makes rapamycin cytotoxic rather than cytostatic at doses that are tolerated clinically. AICAR by itself is able to suppress mTOR complex 1 (mTORC1), but also stimulates a feedback activation of mTORC2, which activates the survival kinase Akt. However, AICAR also suppresses production of phosphatidic acid (PA), which interacts with mTOR in a manner that is competitive with rapamycin. The reduced level of PA sensitizes mTORC2 to rapamycin at tolerable nano-molar doses leading reduced Akt phosphorylation and apoptosis. This study reveals how the use of AICAR enhances the efficacy of rapamycin such that rapamycin at low nano-molar doses can suppress mTORC2 and induce apoptosis in human cancer cells at doses that are clinically tolerable.     

FAK/mTOR信号通在肿瘤细胞中的调控作用

粘附斑激酶（focal adhesion kinase,FAK）是一种胞质非受体酪氨酸激酶,是整合素信号通路里一个重要的调节因子,在肿瘤细胞中高表达,与细胞迁移、粘附和侵袭有关。mTOR (mammalian target of rapamycin)是非典型性的Ser/Thr激酶,属于PIKK（phosphatidylinositol kinase related kinase）超家族,介导营养信号调控细胞生长、分化及代谢等生理过程。近年的研究发现FAK通过三条途径与mTOR相关联,组成FAK/mTOR信号通路,在肿瘤细胞的增殖、迁移及肿瘤微环境中发挥着重要的调控作用。本文综述了FAK、mTOR和FAK/mTOR信号通路的特点及对肿瘤细胞调控作用的研究概况,为肿瘤治疗提供参考。     

Pharmacological Manipulation of the Akt Signaling Pathway Regulates Myxoma Virus Replication and Tropism in Human Cancer Cells

Viruses have evolved an assortment of mechanisms for regulating the Akt signaling pathway to establish a cellular environment more favorable for viral replication. Myxoma virus (MYXV) is a rabbit-specific poxvirus that encodes many immunomodulatory factors, including an ankyrin repeat-containing host range protein termed M-T5 that functions to regulate tropism of MYXV for rabbit lymphocytes and certain human cancer cells. MYXV permissiveness in these human cancer cells is dependent upon the direct interaction between M-T5 and Akt, which has been shown to induce the kinase activity of Akt. In this study, an array of compounds that selectively manipulate Akt signaling was screened and we show that only a subset of Akt inhibitors significantly decreased the ability of MYXV to replicate in previously permissive human cancer cells. Furthermore, reduced viral replication efficiency was correlated with lower levels of phosphorylated Akt. In contrast, the PP2A-specific phosphatase inhibitor okadaic acid promoted increased Akt kinase activation and rescued MYXV replication in human cancer cells that did not previously support viral replication. Finally, phosphorylation of Akt at residue Thr308 was shown to dictate the physical interaction between Akt and M-T5, which then leads to phosphorylation of Ser473 and permits productive MYXV replication in these human cancer cells. The results of this study further characterize the mechanism by which M-T5 exploits the Akt signaling cascade and affirms this interaction as a major tropism determinant that regulates the replication efficiency of MYXV in human cancer cells.Following viral infection, substantial alterations in cellular physiology often lead to modification of various cellular pathways critical to the success of viral replication. The demands for energy, nutrients, and macromolecular synthesis that accompany viral replication can be substantial; thus, many viruses have evolved elaborate strategies for hijacking key cellular signaling networks necessary to support their demands (9). By the same token, antiviral pathways activated by the virus infection may also need to be blocked or subverted to ensure successful virus replication. Poxviruses possess large double-stranded DNA (dsDNA) genomes that encode multiple gene products that specifically modify or debilitate the various host signaling responses of the infected cell (28). Many of the immunoregulatory factors expressed by poxviruses have been well characterized, and these factors include virokines, viroreceptors, signaling modulators, and inhibitors of various antiviral responses, such as initiation of apoptosis pathways and signaling by protective cytokines, like interferon and tumor necrosis factor (TNF) (42).Myxoma virus (MYXV) is a member of the Leporipoxvirus genus and exhibits a restricted pathogenesis that is limited to rabbits, primarily due to its specific immunomodulation of the immune system of leporids (48). In rabbits (Sylvilagus spp.) of the Americas, MYXV infection results in a benign infection, characterized by a cutaneous fibroma restricted to the site of inoculation (14); however, the same virus causes a rapid systemic and highly lethal infection called myxomatosis in European rabbits (Oryctolagus cuniculus) (15). Although MYXV has a narrow host range in nature and is pathogenic only to European rabbits, the tropism of MYXV has recently been extended to include human tumor cells in vitro (6, 47, 54, 57, 60) and in xenografted mice in vivo (24, 25, 61). The mechanisms that mediate MYXV tropism in human cancer cells are still being investigated, but one signaling requirement has been linked to the state of cellular Akt kinase activity (57). Human cancer cells (called type I) that exhibit high levels of endogenous phosphorylated Akt (Ser473 and Thr308) supported permissive MYXV replication, while cells with no detectable endogenous phosphorylated Akt, which were unaffected by the virus infection, were nonpermissive (type III). A unique subset of cancer cells (type II) were found to be permissive to wild-type MYXV but did not support MYXV replication following the deletion of the viral host range factor M-T5 (vMyxT5KO). These type II cells constitutively expressed only low levels of endogenous phosphorylated Akt (mostly at Thr308), but following infection with permissive MYXV, a significant increase in Akt phosphorylation (particularly at Ser473) was observed. In stark contrast, the endogenous levels of phosphorylated Akt remained essentially unchanged when type II cells were infected with the nonpermissive M-T5 knockout virus MYXV (vMyxT5KO) (57).The host range factor M-T5 is essential for MYXV replication in rabbit primary lymphocytes (RL-5 cells) and for virus pathogenesis in European rabbits (31). Structurally, M-T5 possesses seven ankyrin (ANK) repeats and a carboxyl-terminal PRANC (pox protein repeats of ankyrin C-terminal) motif, which closely resembles a cellular protein motif called the F-box domain (29). Interaction between M-T5 and components of the cellular SCF (Skp-cullin-F-box) ubiquitin ligase complex was shown to protect MYXV-infected cells from cell cycle arrest (19). In MYXV-infected type II human cancer cells, physical interaction between M-T5 and cellular Akt was shown to upregulate the kinase activity of Akt (57). In another study, M-T5 was shown to be functionally interchangeable with the host ANK repeat-containing protein PIKE-A, and activation of Akt by either PIKE-A or the viral M-T5 protein was sufficient to mediate MYXV permissiveness in type II human cancer cells (59). Similarly, addition of the immunosuppressant drug rapamycin was successful at rescuing vMyxT5KO replication in type II cells by upregulating Akt activation through the mTOR pathway (47). The critical role of Akt in the regulation of multiple biological processes makes Akt a central regulator of cellular signaling, and therefore, it is not surprising that many viruses have developed sophisticated strategies for manipulating the activation of Akt (9, 11).The serine/threonine kinase Akt (also called protein kinase B [PKB]) was initially discovered as the cellular homolog of the viral oncogene (v-Akt) carried by the AKT8 retrovirus isolated from a murine T-cell lymphoma (7, 20, 46). There are three isoforms found in mammals (Akt1, -2, and -3), encoded by separate genes but sharing over 80% amino acid sequence identity. Activation of Akt is predominantly dependent upon phosphoinositide 3-kinase (PI3K), which phosphorylates phosphoinositides (PIs) at the D3 position of the inositol ring to generate PI(3,4,5)P₃ (PIP₃). Akt possesses an N-terminal PH (pleckstrin homology) domain that binds PIP₃ to promote its translocation of the plasma membrane. Once localized at the membrane, Akt becomes phosphorylated at residue Thr308 in the activation loop by phosphoinositide-dependent kinase 1 (PDK1) and also within the carboxy terminus at residue Ser473 by mTORC2 (mammalian target of rapamycin complex 2) (2, 49, 50). Phosphorylation of both sites is necessary for full induction of Akt kinase activity. Akt is a key regulator of many important cellular functions, including cell survival, proliferation, glucose metabolism, and protein synthesis. In the majority of human cancer cells, the Akt pathway is either mutated or constitutively activated, contributing to cancer progression through both stimulation of cellular proliferation and inhibition of apoptosis (34, 55).In this study, we screened an array of Akt inhibitor compounds that selectively manipulate the Akt signaling network at some level and report that certain Akt inhibitors significantly blocked MYXV replication in previously permissive type I and II human cancer cells. An additional set of inhibitors selectively inhibited only the replication of MYXV deleted for M-T5 and did not modify the replicative ability of the parental wild-type virus. Furthermore, the decrease in viral replication efficiency was correlated with lower levels of phosphorylated Akt at residues Thr308 and Ser473. In contrast, certain PP2A-specific phosphatase inhibitors, such as okadaic acid, promoted increased Akt kinase activation and rescued MYXV replication in type III human cancer cells that did not previously support viral replication. Finally, we demonstrate that the hemi-phosphorylation of Akt at residue Thr308 dictates physical interaction between Akt and M-T5, which ultimately leads to productive MYXV replication in type II cancer cells. These studies show that activation of the Akt signaling cascade is essential for efficient MYXV replication in human cancer cells and further demonstrate the dynamic role by which M-T5 manipulates Akt signaling to establish a cellular environment more favorable for viral replication.     

Oncolytic Properties of a Mumps Virus Vaccine Strain in Human Melanoma Cell Lines

The oncolytic potential of the attenuated mumps virus (MV) vaccine strain Leningrad-3 (L-3) was evaluated in a panel of four human metastatic melanoma cell lines. The lines were shown to be susceptible and permissive to MV infection. Efficient MV replication led to death of melanoma cells, but the effect differed among the cell lines. Possible mechanisms mediating the selectivity of MV L-3 towards the cell lines were explored. Replicative and oncolytic activity of MV was found to depend on the expression pattern of type I interferon genes. None of the melanoma cell lines showed induction of expression of the total spectrum of genes required to inhibit virus replication. Based on the results, MV L-3 was assumed to be a promising oncolytic agent for human melanoma cells.     

p21-activated kinase 1 is activated through the mammalian target of rapamycin/p70 S6 kinase pathway and regulates the replication of hepatitis C virus in human hepatoma cells

Cellular mechanisms that regulate the replication of hepatitis C virus (HCV) RNA are poorly understood. p21-activated kinase 1 (PAK1) is a serine/threonine kinase that has been suggested to participate in antiviral signaling. We studied its role in the cellular control of HCV replication. Transfection of PAK1-specific small interfering RNA enhanced viral RNA and protein abundance in established replicon cell lines as well as cells infected with chimeric genotype 1a/2a HCV, despite reducing cellular proliferation, suggesting specific regulation of HCV replication. PAK1 knockdown did not reduce interferon regulatory factor 3-dependent gene expression, indicating that this regulation is independent of the retinoic acid-inducible gene I/interferon regulatory factor 3 pathway. On the other hand, LY294002 and rapamycin abolished PAK1 phosphorylation and enhanced HCV abundance, suggesting that the mammalian target of rapamycin (mTOR) is involved in PAK1 regulation of HCV. Small interfering RNA knockdown of the mTOR substrate p70 S6 kinase abrogated PAK1 phosphorylation and enhanced HCV RNA abundance, whereas overexpression of a constitutively active alternate substrate, eukaryotic translation initiation factor 4E-binding protein 1, increased cap-independent viral translation and viral RNA abundance without influencing PAK1 phosphorylation. Similar data indicated that mTOR is regulated by both phosphatidylinositol 3-kinase/Akt and ERK. Taken together, the data indicate that p70 S6 kinase activates PAK1 and contributes to phosphatidylinositol 3-kinase- and ERK-mediated regulation of HCV RNA replication.     

TOR kinase and Ran are downstream from PI3K/Akt in H2O2-induced mitosis

Hydrogen peroxide (H2O2) activates signaling cascades essential for cell proliferation via phosphatidylinositol-3-kinase (PI3K) and Akt. Here we show that induction of mitogenic signaling by H2O2 activates sequentially PI3K, Akt, mammalian target of rapamycin (mTOR), and Ran protein. Akt activation is followed by signaling through the mTOR kinase and upregulation of Ran in primary type II pneumocytes, a cell type implicated in the development of lung adenocarcinoma. Pretreatment of the cells with wortmannin, a specific inhibitor of PI3K, or rapamycin, a specific inhibitor of mTOR kinase, prevented H2O2-increased mitosis. H2O2-induced Akt ser-473 phosphorylation and upregulation of Ran protein were prevented by wortmannin but not by rapamycin, indicating that PI3K is upstream of Akt and mTOR is downstream from Akt. Overexpression of myr-Akt or Ran-wt in type II pneumocytes increased Akt ser-473 phosphorylation and mitosis in a catalase-dependent manner, indicating that H2O2 is essential for Akt and Ran signaling. These results indicate that H2O2-induced mitogenic signaling in primary type II pneumocytes is mediated by PI3K, Akt, mTOR-kinase, and Ran protein.     

Participation of mTOR in the regulation of multidrug resistance of tumor cells

Molecular mechanisms of the influence of PI3K/Akt/PTEN/mTOR-signaling pathway on survival of tumor cells treated with cytotoxic drugs was studied using rapamycin (Rapa), mTOR specific inhibitor, and 9 human tumor cell lines of different origin and with different Akt kinase activity. Three of these cell lines were selected for drug resistance due to P-glycoprotein (Pgp or ABCB1) overexpression. Rapa inhibited phosphorylation of mTOR downstream effectors. Rapa sensitivity of the cells was Akt-dependent but did not correlate with ABCB1 overexpression. Suppression of mTOR function increased drug resistance in 8 out of 9 cell lines studied. The influence of Rapa on the ABC-transporter gene expression was examined. It was shown that in half of the cell lines studied Rapa exerted differential effects on the amount of ABC-transporter proteins: in some cases the protein amount decreased and in others, increased. The amount of mRNA remained unchanged. These data suggest that mTOR can regulate ABC transporters at the level of translation.     

High-dose rapamycin induces apoptosis in human cancer cells by dissociating mTOR complex 1 and suppressing phosphorylation of 4E-BP1

mTOR, the mammalian target of rapamycin, has been widely implicated in signals that promote cell cycle progression and survival in cancer cells. Rapamycin, which inhibits mTOR with high specificity, has consequently attracted much attention as an anticancer therapeutic. Rapamycin suppresses phosphorylation of S6 kinase at nanomolar concentrations; however, at higher micro-molar doses, rapamycin induces apoptosis in several human cancer cell lines. While much is known about the effect of low-dose rapamycin treatment, the mechanistic basis for the apoptotic effects of high-dose rapamycin treatment is not understood. We report here that the apoptotic effects of high-dose rapamycin treatment correlate with suppressing phosphorylation of the mTOR complex 1 substrate, eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). Consistent with this observation, ablation of eIF4E also resulted in apoptorsis in MDA-MB 231 breast cancer cells. We also provide evidence that the differential dose effects of rapamycin are correlated with partial and complete dissociation of Raptor from mTORC1 at low and high doses, respectively. In contrast with MDA-MB-231 cells, MCF-7 breast cancer cells survived rapamycin-induced suppression of 4E-BP1 phosphorylation. We show that survival correlated with a hyperphosphorylation of Akt at S473 at high rapamycin doses, the suppression of which conferred rapamycin sensitivity. This study reveals that the apoptotic effect of rapamycin requires doses that completely dissociate Raptor from mTORC1 and suppress that phosphorylation of 4E-BP1 and inhibit eIF4E.Key words: rapamycin, mTOR, 4E-BP1, eIF4E, Akt, apoptosis     

The mTOR (mammalian target of rapamycin) kinase maintains integrity of mTOR complex 2

In higher eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Deregulation of PI3K/Akt signaling is linked to human diseases, including cancer and metabolic disorders. The PI3K-dependent signaling kinase complex mTORC2 (mammalian target of rapamycin complex 2) has been defined as the regulatory Ser-473 kinase of Akt. The regulation of mTORC2 remains very poorly characterized. We have reconstituted mTORC2 by its assembly in vitro or by co-expression its four essential components (rictor, SIN1, mTOR, mLST8). We show that the functional mTOR kinase domain is required for the mTORC2 activity as the Ser-473 kinase of Akt. We also found that mTOR by phosphorylation of SIN1 prevents its lysosomal degradation. Thus, the kinase domain of mTOR is required for the functional activity of mTORC2, and it controls integrity of mTORC2 by maintaining the protein stability of SIN1.     

Vesicular stomatitis virus oncolysis of T lymphocytes requires cell cycle entry and translation initiation

Vesicular stomatitis virus (VSV) is a candidate oncolytic virus that replicates and induces cell death in cancer cells while sparing normal cells. Although defects in the interferon antiviral response facilitate VSV oncolysis, other host factors, including translational and growth regulatory mechanisms, also appear to influence oncolytic virus activity. We previously demonstrated that VSV infection induces apoptosis in proliferating CD4(+) T lymphocytes from adult T-cell leukemia samples but not in resting T lymphocytes or primary chronic lymphocytic leukemia cells that remain arrested in G(0). Activation of primary CD4(+) T lymphocytes with anti-CD3/CD28 is sufficient to induce VSV replication and cell death in a manner dependent on activation of the MEK1/2, c-Jun NH(2)-terminal kinase, or phosphatidylinositol 3-kinase pathway but not p38. VSV replication is specifically impaired by the cell cycle inhibitor olomoucine or rapamycin, which induces early G(1) arrest, but not by aphidicolin or Taxol, which blocks at the G(1)1S or G(2)1M phase, respectively; this result suggests a requirement for cell cycle entry for efficient VSV replication. The relationship between increased protein translation following G(0)/G(1) transition and VSV permissiveness is highlighted by the absence of mTOR and/or eIF4E phosphorylation whenever VSV replication is impaired. Furthermore, VSV protein production in activated T cells is diminished by small interfering RNA-mediated eIF4E knockdown. These results demonstrate that VSV replication in primary T lymphocytes relies on cell cycle transition from the G(0) phase to the G(1) phase, which is characterized by a sharp increase in ribogenesis and protein synthesis.