Enhancing therapeutic efficacy by targeting non-oncogene addicted cells with combinations of signal transduction inhibitors and chemotherapy.

The effects of inhibition of the Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways and chemotherapeutic drugs on cell cycle progression and drug sensitivity were examined in cytokine-dependent FL5.12 hematopoietic cells. We examined their effects, as these cells resemble normal hematopoietic precursor cells as they do not exhibit "oncogene-addicted" growth, while they do display "cytokine-addicted" proliferation as cytokine removal resulted in apoptosis in greater than 80% of the cells within 48 hrs. When cytokine-dependent FL5.12 cells were cultured in the presence of IL-3, which stimulated multiple proliferation and anti-apoptotic cascades, MEK, PI3K and mTOR inhibitors transiently suppressed but did not totally inhibit cell cycle progression or induce apoptosis while chemotherapeutic drugs such as doxorubicin and paclitaxel were more effective in inducing cell cycle arrest and apoptosis. Doxorubicin induced a G(1) block, while paclitaxel triggered a G(2)/M block. Doxorubicin was more effective in inducing cell death than paclitaxel. Furthermore the effects of doxorubicin could be enhanced by addition of MEK, PI3K or mTOR inhibitors. Cytokine-dependent cells which proliferate in vitro and are not "oncogene-addicted" may represent a pre-malignant stage, more refractory to treatment with targeted therapy. However, these cells are sensitive to chemotherapeutic drugs. It is important to develop methods to inhibit the growth of such cytokine-dependent cells as they may resemble the leukemia stem cell and other cancer initiating cells. These results demonstrate the enhanced effectiveness of targeting early hematopoietic progenitor cells with combinations of chemotherapeutic drugs and signal transduction inhibitors.

The Raf/MEK/ERK pathway can govern drug resistance, apoptosis and sensitivity to targeted therapy.

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on proliferation, drug resistance, prevention of apoptosis and sensitivity to signal transduction inhibitors were examined in FL/DeltaAkt-1:ER*(Myr(+)) + DeltaRaf-1:AR cells which are conditionally-transformed to grow in response to Raf and Akt activation. Drug resistant cells were isolated from FL/DeltaAkt-1:ER*(Myr(+)) + DeltaRaf-1:AR cells in the presence of doxorubicin. Activation of Raf-1, in the drug resistant FL/DeltaAkt-1:ER*(Myr(+)) + DeltaRaf-1:AR cells, increased the IC(50) for doxorubicin 80-fold, whereas activation of Akt-1, by itself, had no effect on the doxorubicin IC50. However, Akt-1 activation enhanced cell proliferation and clonogenicity in the presence of chemotherapeutic drugs. Thus the Raf/MEK/ERK pathway had profound effects on the sensitivity to chemotherapeutic drugs, and Akt-1 activation was required for the long term growth of these cells as well as resistance to chemotherapeutic drugs. The effects of doxorubicin on the induction of apoptosis in the drug resistant cells were enhanced by addition of either mTOR and MEK inhibitors. These results indicate that targeting the Raf/MEK/ERK and PI3K/Akt/mTOR pathways may be an effective approach for therapeutic intervention in drug resistant cancers that have mutations activating these cascades.

Dominant roles of the Raf/MEK/ERK pathway in cell cycle progression, prevention of apoptosis and sensitivity to chemotherapeutic drugs.

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on cell cycle progression, gene expression, prevention of apoptosis and sensitivity to chemotherapeutic drugs were examined in FL/ΔAkt-1:ER*(Myr(+)) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf-1 and Akt-1 activation by treatment with testosterone or tamoxifen respectively. In these cells we can compare the effects of normal cytokine vs. oncogene mediated signaling in the same cells by changing the culture conditions. Raf-1 was more effective than Akt-1 in inducing cell cycle progression and preventing apoptosis in the presence and absence of chemotherapeutic drugs. The normal cytokine for these cells, interleukin-3 induced/activated most downstream genes transiently, with the exception of p70S6K that was induced for prolonged periods of time. In contrast, most of the downstream genes induced by either the activate Raf-1 or Akt-1 oncogenes were induced for prolonged periods of time, documenting the differences between cytokine and oncogene mediated gene induction which has important therapeutic consequences. The FL/ΔAkt-1:ER*(Myr(+)) + ΔRaf-1:AR cells were sensitive to MEK and PI3K/mTOR inhibitors. Combining MEK and PI3K/mTOR inhibitors increased the induction of apoptosis. The effects of doxorubicin on the induction of apoptosis could be enhanced with MEK, PI3K and mTOR inhibitors. Targeting the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be an effective approach for therapeutic intervention in those cancers which have upstream mutations which result in activation of these pathways.

Development of a conditional in vivo model to evaluate the efficacy of small molecule inhibitors for the treatment of Raf-transformed hematopoietic cells.

Conditionally active forms of the Raf proteins (Raf-1, B-Raf, and A-Raf) were created by ligating NH2-terminal truncated activated forms (Delta) to the estrogen receptor (ER) hormone-binding domain resulting in estradiol-regulated constructs (DeltaRaf:ER). These different Raf:ER oncoproteins were introduced into the murine FDC-P1 hematopoietic cell line, and cells that grew in response to the three DeltaRaf:ER oncoproteins were isolated. The ability of FDC-P1, DeltaRaf-1:ER, DeltaA-Raf:ER, and DeltaB-Raf:ER cells to form tumors in severe combined immunodeficient mice was compared. Mice injected with DeltaRaf:ER cells were implanted with beta-estradiol pellets to induce the DeltaRaf:ER oncoprotein. Cytokine-dependent parental cell lines did not form tumors. Implantation of beta-estradiol pellets into mice injected with DeltaRaf:ER cells significantly accelerated tumor onset and tumor size. The recovered DeltaRaf:ER cells displayed induction of extracellular signal-regulated kinase (ERK) in response to beta-estradiol stimulation, indicating that they had retained conditional activation of ERK even when passed through a severe combined immunodeficient mouse. The DeltaRaf:ER cells were very sensitive to induction of apoptosis by the mitogen-activated protein/ERK kinase (MEK) 1 inhibitor CI1040 whereas parental cells were much less affected, demonstrating that the MEK1 may be useful in eliminating Ras/Raf/MEK-transformed cells. Furthermore, the effects of in vivo administration of the MEK1 inhibitor were evaluated and this inhibitor was observed to suppress the tumorigenicity of the injected cells. This DeltaRaf:ER system can serve as a preclinical model to evaluate the effects of signal transduction inhibitors which target the Raf and MEK proteins.

The epidermal growth factor receptor gene family as a target for therapeutic intervention in numerous cancers: what's genetics got to do with it?

Over the past 30 years, a relatively simple growth factor and its cognate receptor have provided seminal insights into the understanding of the genetic basis of cancer, as well as growth factor signalling. The epidermal growth factor (EGF), its cognate receptor (EGFR) and related family members have been shown to be important in normal, as well as the malignant growth of many cell types including: glioblastomata, astrocytomas, medulloblastomata, non-small cell lung carcinoma (NSCLC) and breast cancer. This review summarises the history of the EGFR gene and the v-ErbB oncogene, as well as diverse approaches developed to inhibit EGFR activity. The two most advanced therapies use either small-molecule cell membrane permeable kinase inhibitors or antibodies which prevent receptor activation. Recent clinical trials indicate that certain NSCLC patients have mutations in the EGFR gene which makes them more responsive to kinase inhibitors. These mutations appear to enhance the ability of the ligand to activate EGFR activity and also prolong the binding of the EGFR inhibitor to the kinase domain. Evidence to date suggests that these EGFR mutations in NSCLC occur more frequently in Japan than in the western hemisphere. Although these mutations are correlated with enhanced efficacy to the inhibitors in NSCLC, they can not explain or predict the sensitivity of many other cancer patients to the beneficial effects of the EGFR kinase inhibitors or antibody mediated therapy. As with as other small-molecule kinase inhibitors and susceptible diseases (e.g., imatinib and chronic myeloid leukaemia), resistance to EGFR inhibitors has been reported recently, documenting the requirement for development of multi-pronged therapeutic approaches. EGFR kinase inhibitors are also being evaluated as adjuvants in hormonal therapy of breast cancer - especially those which overexpress EGFR. Genetically engineered antibodies specific for the EGFR family member ErbB2 have been developed which show efficacy in the treatment of primary, and prevent the relapse of, breast cancer. Clearly, the EGF/EGFR signalling cascade has, and continues to play, an important role in the development of novel anticancer targeted therapies.

Effects of endogenous epidermal growth factor receptor signaling on DNA synthesis and ERK activation in a cytokine-dependent hematopoietic cell line.

Epidermal growth factor (EGF) is commonly thought to affect the proliferation of many cells, especially epithelial cells. Aberrant expression of the receptor for EGF, (EGFR) or members of the EGFR family is often implicated in the etiology of many cancers. Ligation of the EGFR results in the activation of many downstream signaling pathways which have profound effects on cell cycle progression and the prevention of apoptosis. In general, the EGFR is thought to be either not expressed or expressed at low levels in hematopoietic cells. We determined that the EGFR was expressed at a low level in the murine cytokine-dependent hematopoietic cell line FDC-P1 but not in an additional murine IL-3 dependent cell line FL5. EGF induced a mild effect on DNA synthesis and ERK activation in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Addition of suboptimal concentrations of IL-3 synergized with EGF in stimulating DNA synthesis in EGFR-positive FDC-P1 cells. Likewise, the EGFR inhibitor AG1478 induced apoptosis in EGFR positive FDC-P1 cells but not EGFR negative FL5.12 cells. Both cell lines can be directly transformed to cytokine independence by activated EGFR (v-ERBB) expression in the absence of autocrine growth factors indicating that they are poised to fully utilize EGFR mediated signal transduction pathways as a means for proliferation. These results document the functional importance of endogenous EGFR signaling pathway in some hematopoietic cells.

Conditional EGFR promotes cell cycle progression and prevention of apoptosis in the absence of autocrine cytokines.

v-ErbB is an oncogene related to the Epidermal Growth Factor Receptor (EGFR). EGFR overexpression has been observed in many pathological situations. There is a truncated form of EGFR, referred to as EGFvIII, which resembles v-ErbB in biological properties and is often expressed in certain human tumors. Aberrant EGFR expression in human cancers is often constitutive and may occur in the presence of mutated oncogenes or tumor suppressor genes. To circumvent these problems, we subcloned v-ErbB into a vector which contains the estrogen receptor hormone binding domain (ER) which renders the v-ErbB:ER protein dependent upon beta-estradiol for activity. v-ErbB:ER conditionally abrogated the cytokine dependence of hematopoietic cells more efficiently than activated v-Ha-Ras, v-Src, Raf or Akt. Abrogation of cytokine-dependence by v-ErbB:ER was not due to the synthesis of autocrine growth factors. Treatment of v-ErbB:ER cells with the EGFR inhibitor AG1478 efficiently induced apoptosis. Induction of apoptosis and prevention of cell cycle progression by the EGFR inhibitor were only observed when the cells were grown in response to v-ErbB:ER activation demonstrating specificity. In contrast, the other inhibitors suppressed cell cycle progression when the cells were grown in response to v-ErbB:ER or the cytokine interleukin-3. When MEK and either EGFR or PI3K/mTOR inhibitors were added, an enhanced apoptotic response was observed. Thus this conditional ErbB construct is useful to elucidate EGFR signaling and anti-apoptotic pathways in the absence of autocrine cytokine expression.

Solubility and bioactivity of the Pseudomonas quinolone signal are increased by a Pseudomonas aeruginosa-produced surfactant.

Pseudomonas aeruginosa is a gram-negative bacterium that causes serious infections in immunocompromised individuals and cystic fibrosis patients. This opportunistic pathogen controls many of its virulence factors and cellular functions through the activity of three cell-to-cell signals, N-(3-oxododecanoyl)-L-homoserine lactone, N-butyryl-L-homoserine lactone, and the Pseudomonas quinolone signal (PQS). The activity of these signals is dependent upon their ability to dissolve in and freely diffuse through the aqueous solution in which P. aeruginosa happens to reside. Despite this, our data indicated that PQS was relatively insoluble in aqueous solutions, which led us to postulate that P. aeruginosa could be producing a PQS-solubilizing factor. In this report, we show that the P. aeruginosa-produced biosurfactant rhamnolipid greatly enhances the solubility of PQS in aqueous solutions. The enhanced solubility of PQS led to an increase in PQS bioactivity, as measured by both a gene induction assay and an apoptosis assay. This is the first demonstration of the importance of a bacterial surfactant in the solubilization and bioactivity of a cell-to-cell signal.

Effects of a conditionally active v-ErbB and an EGF-R inhibitor on transformation of NIH-3T3 cells and abrogation of cytokine dependency of hematopoietic cells.

Epidermal growth factor (EGF) and its cognate receptor (EGF-R) are often dysregulated in human neoplasia. Moreover, EGF-R-transformed cell lines have constitutive EGF-R activity, which makes elucidation of its effects difficult to determine. In the following studies, the effects of a novel conditionally activated form of EGF-R, v-ErbB:ER, on the morphological transformation of NIH-3T3 cells and the abrogation of hematopoietic cell cytokine dependence were investigated. The v-ErbB ES-4 oncogene was fused to the hormone binding domain of the estrogen receptor (ER). This construct, v-ErbB:ER, requires beta-estradiol or 4-OH tamoxifen for activation. v-ErbB:ER conditionally transformed NIH-3T3 cells and abrogated cytokine dependence of hematopoietic cells. Stimulation of v-ErbB:ER activity resulted in the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and Raf/MEK/ERK kinase cascades. To determine the importance of these signal transduction pathways, the conditionally transformed hematopoietic cells were treated with EGF-R, PI3K and MEK inhibitors. The EGF-R inhibitor AG1478 effectively inhibited MEK, ERK and Akt activation, and induced apoptosis when the cells were grown in response to v-ErbB:ER. Apoptosis was observed at 100- to 1000-fold lower concentrations of AG1478 when the cells were grown in response to v-ErbB:ER as opposed to IL-3. Furthermore, the parental, BCR-ABL- and Raf-transformed cells were only susceptible to the apoptosis-inducing effects of AG1478 at the highest concentrations demonstrating the specificity of these inhibitors. MEK or PI3K inhibitors suppressed ERK or Akt activation, respectively, and induced apoptosis in the v-ErbB:ER-responsive cells. However, MEK and PI3K inhibitors only induced apoptosis at 1000-fold higher concentrations than the EGFR inhibitor. This novel v-ErbB:ER construct and these conditionally transformed cell lines will be useful to further elucidate ErbB-mediated signal transduction and to determine the effectiveness of various inhibitors in targeting different aspects of EGF-R-mediated signal transduction and malignant transformation.

Raf promotes human herpesvirus-8 (HHV-8/KSHV) infection.

Human herpesvirus-8 (HHV-8/KSHV) is etiologically associated with Kaposi's sarcoma (KS) and other tumors. Constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway has been associated with a variety of tumors, including AIDS-related KS. The oncoprotein Raf is situated at a pivotal position in regulating the MAPK pathway. Hence, we analysed the effect of oncoprotein Raf on HHV-8 infectious entry into target cells. Here we report Raf expression to significantly enhance HHV-8 infection of target cells. These findings implicate a role for Raf not only in the infectious entry of HHV-8 but also in modulating KS pathogenesis.

Ability of the activated PI3K/Akt oncoproteins to synergize with MEK1 and induce cell cycle progression and abrogate the cytokine-dependence of hematopoietic cells.

Multiple signal transduction pathways, including the Raf/MEK/ERK and PI3K/Akt kinase cascades, play critical roles in transducing growth signals from activated cell surface receptors. Using conditionally and constitutively-active forms of MEK1 and either PI3K or Akt, we demonstrate synergy between these kinases in relieving cytokine-dependence of the FDC-P1 hematopoietic cell line. Cytokine-independent cells were obtained from DeltaMEK1:ER-infected cells at a frequency of 5 x 10(-5) indicating that low frequency of cells expressing beta-estradiol-regulated DeltaMEK1:ER became factor-independent, while activated PI3K or Akt by themselves did not relieve cytokine-dependence. In contrast, cytokine-independent cells were recovered approximately 25 to 250-fold more frequently from DeltaMEK1:ER infected cells also infected with either activated PI3K or Akt. MEK/PI3K and MEK/Akt-responsive cells could be maintained long-term as long as either beta-estradiol or the estrogen receptor antagonist 4-hydroxy-tamoxifen (4HT) were provided. The MEK/PI3K/Akt responsive cells were sensitive to both MEK and PI3K/Akt/p70S6K inhibitors. Synergy was observed when inhibitors which targeted both pathways were added together. These results indicate that there is synergy between the Raf/MEK/ERK and PI3K/Akt pathways in terms of abrogation of cytokine-dependence of hematopoietic cells. Likewise, suppression of multiple signal transduction pathways is a more effective means to inhibit cell cycle progression and induce apoptosis in leukemic cells.

B-raf and insulin synergistically prevent apoptosis and induce cell cycle progression in hematopoietic cells.

FDC-P1 hematopoietic cells were conditionally transformed to grow in response to (delta)B Raf:ER, (delta)Raf-1:ER or DA-Raf:ER in which the hormone binding domain of the estrogen receptor (ER) was linked to the N-terminal truncated (delta) Raf genes. When these cells were deprived of IL-3 or beta-estradiol for 24 hrs, they exited the cell cycle and underwent apoptosis. FD/(delta)Raf-1:ER and FD/(delta)A-Raf:ER, but not FD/(delta)B-Raf:ER cells, were readily induced to re-enter the cell cycle after addition of beta-estradiol or IL-3. Deprived FD/(delta)Raf-1:ER, but not FD/(delta)B-Raf:ER cells, expressed activated forms of MEK1 and ERK after beta-estradiol or IL-3 stimulation. Insulin or beta-estradiol alone did not induce FD/(delta)B-Raf:ER cells to re-enter the cell cycle, whereas cell cycle entry was observed upon their co-addition. Apoptosis was prevented in FD/(delta)B-Raf:ER cells when they were cultured in the presence of IL-3 or beta-estradiol, whereas they underwent apoptosis in their absence. Insulin by itself did not prevent apoptosis, however, upon DB-Raf:ER or DRaf-1:ER activation and addition of insulin, more than an additive effect was observed in both lines indicating that these path- ways synergized to prevent apoptosis. Raf isoforms differ in their abilities to control apoptosis and cell cycle progression and B-Raf requires insulin-activated pathways for full antiapoptotic and proliferative activity.

Synergy between PI3K/Akt and Raf/MEK/ERK pathways in IGF-1R mediated cell cycle progression and prevention of apoptosis in hematopoietic cells.

The insulin like growth factor-1 (IGF-1) receptor (R) induced PI3K/Akt signal transduction cascade has critical roles in prevention of apoptosis and regulation of cell cycle progression. Here, we discuss the effects of IGF-1R-mediated signal transduction on hematopoietic cells which normally require interleukin-3 (IL-3) for growth and prevention of apoptosis. Cytokine-dependent FDC-P1 hematopoietic cells were conditionally transformed to grow in response to overexpression of IGF-1R in the presence of IGF-1. When these cells were deprived of IL-3 or IGF-1 for 24 hrs, they exited the cell cycle, activated caspase 3 and underwent apoptosis. The effects of inhibitors which targeted the PI3K/Akt and Raf/MEK/ERK pathways were determined. When the cells were cultured with IGF-1 and either PI3K or MEK inhibitors, cell cycle progression and DNA synthesis were inhibited and caspase 3 activity and apoptosis were induced. Coinhibition of both pathways synergized to prevent cell cycle progression, inhibit DNA synthesis and induce apoptosis. These inhibitors had more apoptotic inducing effects when the cells were grown in response to IGF-1 than IL-3, indicating that IL-3 can induce additional anti-apoptotic pathways. These results demonstrate that the PI3K/Akt and Raf/MEK/ERK pathways are intimately involved in IGF-1R-mediated cell cycle progression and prevention of apoptosis in hematopoietic cells.

Effects of the RAF/MEK/ERK and PI3K/AKT signal transduction pathways on the abrogation of cytokine-dependence and prevention of apoptosis in hematopoietic cells.

The Raf/MEK/ERK kinase cascade is pivotal in transmitting signals from membrane receptors to transcription factors that control gene expression culminating in the regulation of cell cycle progression. This cascade can prevent cell death through ERK2 and p90(Rsk) activation and phosphorylation of apoptotic and cell cycle regulatory proteins. The PI3K/Akt kinase cascade also controls apoptosis and can phosphorylate many apoptotic and cell cycle regulatory proteins. These pathways are interwoven as Akt can phosphorylate Raf and result in its inactivation, and Raf can be required for the antiapoptotic effects of Akt. In this study, the effects of activated Raf (Raf-1, A-Raf and B-Raf) and PI3K/Akt proteins on abrogation of cytokine dependence in FL5.12 hematopoietic cells were examined. Activated Raf, PI3K or Akt expression, by themselves, did not readily relieve cytokine dependence. The presence of activated Raf and PI3K/Akt increased the isolation of factor-independent cells from 400- to 2500-fold depending upon the particular combination examined. The individual effects of activated Raf and Akt on proliferation, apoptosis and autocrine growth factor synthesis were further examined with hormone-inducible constructs (Delta Raf-1:AR and Delta Akt:ER*(Myr(+)). Activation of either Raf or Akt hindered cell death; however, both proliferation and maximal synthesis of autocrine cytokines were dependent upon activation of both signaling pathways. The effects of small molecular weight inhibitors on DNA synthesis and cytokine gene expression were also examined. The PI3K inhibitor, LY294002, inhibited growth and cytokine gene expression. This effect could be synergistically increased by addition of the MEK inhibitor UO126. These cells will be useful in elucidating the interactions between Raf/MEK/ERK and PI3K/Akt cascades in proliferation, apoptosis, and leukemogenesis, as well as evaluating the efficacy of signal transduction inhibitors that target these cascades.

Fibroblastic, hematopoietic, and hormone responsive epithelial cell lines and culture conditions for elucidation of signal transduction and drug resistance pathways by gene transfer.

Elucidation of signal transduction pathways involved in proliferation, cell cycle progression and the regulation of apoptosis has shown great promise in the treatment of various diseases including neoplastic, inflammatory, autoimmune, immunodeficiency, arthritic and neurodegenerative disorders. By understanding how these signal transduction pathways function, chemotherapeutic targets may be identified which will suppress or eliminate the disease. This information may eventually be translated into therapy, which would either eliminate or safely contain the patient's disease. This chapter will focus on basic tissue culture techniques which are used to elucidate signal transduction pathways. Furthermore, this chapter will provide a general background for understanding how gene transfer techniques can be used to elucidate signal transduction pathways as well as various pitfalls commonly encountered with their usage.

Elucidation of signal transduction pathways by transfection of cells with modified oncogenes.

This chapter will focus on introduction of various wild type (WT) and mutant genes into cells by DNA transfection. Techniques for analysis of the inheritance, expression, and biological effects of the introduced genes will be described. Various strong and weak points about three different techniques of stable gene transfer, including calcium-phosphate DNA precipitation, transfection via liposomes, and transfection via electroporation, will be discussed.

Elucidation of signal transduction pathways by retroviral infection of cells with modified oncogenes.

This chapter will focus on understanding how various wild type (WT), dominant negative (DN), constitutively active (CA), and conditionally active (COND) oncogenes, as well as antisense (AS) genes contained in retroviral vectors may be used to elucidate signal transduction pathways. We will describe methods to introduce these genes into cells and subsequent analysis of inheritance, expression, and biological effects of the genes introduced. Furthermore, we will discuss various strong points about each of these different types of constructs, how they can be used to elucidate signal transduction, apoptotic, and drug resistance pathways as well as various pitfalls commonly encountered with their usage.

Regulation of cell cycle progression and apoptosis by the Ras/Raf/MEK/ERK pathway (Review).

The Ras/Raf/MEK/ERK signal transduction pathway regulates cell cycle progression and apoptosis in diverse types of cells. Mutations in this pathway are often observed in transformed cell lines and frequently linked with human cancers. The Ras/Raf/MEK/ERK pathway can induce events both associated with cell proliferation and cell cycle arrest. The particular course chosen may depend on the strength and the particular Raf gene activated by Ras. This pathway also is involved in maintaining cell survival by modulating the activity of apoptotic molecules including Bad and Bcl-2. This review will discuss the regulation of the Ras/Raf/MEK/ERK pathway and how it modulates cell cycle progression and cell survival.