Heat shock and ceramide have different apoptotic pathways in radiation induced fibrosarcoma (RIF) cells

Heat shock induces various cellular responses including inhibition of protein synthesis, production of heat shock proteins (HSPs) and induction of thermotolerance. The molecular mechanisms of the processes have not been well understood. It has been proposed that ceramide formation during heat shock mediates heat shock induced apoptosis. We examined whether C2-ceramide mimicked the cellular response to heat shock in RIF-1 cells and their thermotolerant derivative TR-RIF-1 cells. Discernible effects between heat shock and C2-ceramide treatments were observed in cellular changes such as total protein synthesis, HSP synthesis, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activity and PARP cleavage. Heat shock immediately inhibited cellular protein synthesis, which was recovered by synthesizing HSPs first and then whole proteins later. Heat shock also activated SAPK/JNK and increased PARP cleavage in dose-dependent manner. Thermotolerant TR-RIF-1 cells responded to heat shock more insensitively than RIF-1 cells. On the other hand, C2-ceramide treatment did not accompany any changes induced by heat shock. No discernible differences between RIF-1 and TR-RIF-1 cells were observed by C2-ceramide treatment. We tried to figure out how C2-ceramide interacts with cellular membrane and found that exogenous C2-ceramide was incorporated into the outer monolayer and flipped into the inner monolayer of human erythrocytes in ATP-dependent manner. However, the rate of C2-ceramide incorporation was similar in control and thermotolerant cells. In summary, thermotolerant cells are resistant to heat shock induced apoptotic signaling but not resistant, rather sensitive to membrane disturbing C2-ceramide mediated apoptosis. These results suggest that heat shock and ceramide have different signal transduction pathways.     

Systemic analysis of heat shock response induced by heat shock and a proteasome inhibitor MG132

The molecular basis of heat shock response (HSR), a cellular defense mechanism against various stresses, is not well understood. In this, the first comprehensive analysis of gene expression changes in response to heat shock and MG132 (a proteasome inhibitor), both of which are known to induce heat shock proteins (Hsps), we compared the responses of normal mouse fibrosarcoma cell line, RIF-1, and its thermotolerant variant cell line, TR-RIF-1 (TR), to the two stresses. The cellular responses we examined included Hsp expressions, cell viability, total protein synthesis patterns, and accumulation of poly-ubiquitinated proteins. We also compared the mRNA expression profiles and kinetics, in the two cell lines exposed to the two stresses, using microarray analysis. In contrast to RIF-1 cells, TR cells resist heat shock caused changes in cell viability and whole-cell protein synthesis. The patterns of total cellular protein synthesis and accumulation of poly-ubiquitinated proteins in the two cell lines were distinct, depending on the stress and the cell line. Microarray analysis revealed that the gene expression pattern of TR cells was faster and more transient than that of RIF-1 cells, in response to heat shock, while both RIF-1 and TR cells showed similar kinetics of mRNA expression in response to MG132. We also found that 2,208 genes were up-regulated more than 2 fold and could sort them into three groups: 1) genes regulated by both heat shock and MG132, (e.g. chaperones); 2) those regulated only by heat shock (e.g. DNA binding proteins including histones); and 3) those regulated only by MG132 (e.g. innate immunity and defense related molecules). This study shows that heat shock and MG132 share some aspects of HSR signaling pathway, at the same time, inducing distinct stress response signaling pathways, triggered by distinct abnormal proteins.     

Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana

Salicylic acid (SA) is reported to protect plants from heat shock (HS), but insufficient is known about its role in thermotolerance or how this relates to SA signaling in pathogen resistance. We tested thermotolerance and expression of pathogenesis-related (PR) and HS proteins (HSPs) in Arabidopsis thaliana genotypes with modified SA signaling: plants with the SA hydroxylase NahG transgene, the nonexpresser of PR proteins (npr1) mutant, and the constitutive expressers of PR proteins (cpr1 and cpr5) mutants. At all growth stages from seeds to 3-week-old plants, we found evidence for SA-dependent signaling in basal thermotolerance (i.e. tolerance of HS without prior heat acclimation). Endogenous SA correlated with basal thermotolerance, with the SA-deficient NahG and SA-accumulating cpr5 genotypes having lowest and highest thermotolerance, respectively. SA promoted thermotolerance during the HS itself and subsequent recovery. Recovery from HS apparently involved an NPR1-dependent pathway but thermotolerance during HS did not. SA reduced electrolyte leakage, indicating that it induced membrane thermoprotection. PR-1 and Hsp17.6 were induced by SA or HS, indicating common factors in pathogen and HS responses. SA-induced Hsp17.6 expression had a different dose-response to PR-1 expression. HS-induced Hsp17.6 protein appeared more slowly in NahG. However, SA only partially induced HSPs. Hsp17.6 induction by HS was more substantial than by SA, and we found no SA effect on Hsp101 expression. All genotypes, including NahG and npr1, were capable of expression of HSPs and acquisition of HS tolerance by prior heat acclimation. Although SA promotes basal thermotolerance, it is not essential for acquired thermotolerance.     

Translational thermotolerance provided by small heat shock proteins is limited to cap-dependent initiation and inhibited by 2-aminopurine

Heat shock results in inhibition of general protein synthesis. In thermotolerant cells, protein synthesis is still rapidly inhibited by heat stress, but protein synthesis recovers faster than in naive heat-shocked cells, a phenomenon known as translational thermotolerance. Here we investigate the effect of overexpressing a single heat shock protein on cap-dependent and cap-independent initiation of translation during recovery from a heat shock. When overexpressing alphaB-crystallin or Hsp27, cap-dependent initiation of translation was protected but no effect was seen on cap-independent initiation of translation. When Hsp70 was overexpressed however, both cap-dependent and -independent translation were protected. This finding indicates a difference in the mechanism of protection mediated by small or large heat shock proteins. Phosphorylation of alphaB-crystallin and Hsp27 is known to significantly decrease their chaperone activity; therefore, we tested phosphorylation mutants of these proteins in this system. AlphaB-crystallin needs to be in its non-phosphorylated state to give protection, whereas phosphorylated Hsp27 is more potent in protection than the unphosphorylatable form. This indicates that chaperone activity is not a prerequisite for protection of translation by small heat shock proteins after heat shock. Furthermore, we show that in the presence of 2-aminopurine, an inhibitor of kinases, among which is double-stranded RNA-activated kinase, the protective effect of overexpressing alphaB-crystallin is abolished. The synthesis of the endogenous Hsps induced by the heat shock to test for thermotolerance is also blocked by 2-aminopurine. Most likely the protective effect of alphaB-crystallin requires synthesis of the endogenous heat shock proteins. Translational thermotolerance would then be a co-operative effect of different heat shock proteins.     

Effect of amino acid analogs on the development of thermotolerance and on thermotolerant cells

Exposure of HA-1 Chinese hamster fibroblasts to amino acid analogs has been shown to have a heat-sensitizing effect as well as inducing the heat shock response (Li and Laszlo, 1985a). In this study, we have examined the effect of amino acid analogs on the development of thermotolerance after a brief heat shock or exposure to sodium arsenite and the effect of amino acid analogs on cells that are already thermotolerant. Exposure of HA-1 cells to amino acid analogs inhibited the development of thermotolerance following a mild heat shock or treatment with sodium arsenite. However, cells that were already thermotolerant were resistant to the sensitizing action of amino acid analogs. The refractoriness of thermotolerant cells to amino acid analog treatment developed in parallel with thermotolerance. The uptake of the arginine analog, canavanine, and its incorporation into proteins was not altered in the thermotolerant cells. Furthermore, another biological consequence of exposure to amino acid analogs, sensitization to ionizing radiation, also was not altered in the thermotolerant cells. The inhibition of the development of thermotolerance by amino acid analogs and the refractoriness of thermotolerant cells to the heat-sensitizing action of amino acid analogs lend further support the role of heat-shock proteins in the phenomenon of thermotolerance. © 1993 Wiley-Liss, Inc.     

Involvement of rRNA synthesis in the enhanced survival and recovery of protein synthesis seen in thermotolerance

Although acquired thermotolerance has been linked to the induction of heat shock proteins, the molecular mechanism(s) by which cells become resistant to heat is unknown. The present study shows a strong correlation between the survival of cells following heat shock and the rate of recovery of protein, total RNA, and rRNA synthesis. Increasing exposure of CHO cells to 45 degrees C was found to decrease survival and cause a lengthening delay in these synthetic processes. The same reciprocal correlation was seen in thermotolerant cells. As thermotolerance develops, more cells survive a heat challenge and the delay in synthesis decreases. These data argue that enhanced recovery of protein and RNA synthesis is one factor which plays a key role in thermotolerance. The involvement of rRNA synthesis was further investigated by using actinomycin D at 0.1 microgram m1(-1), a concentration at which rRNA synthesis is selectively inhibited. When the drug was present during the recovery from a challenge heat treatment, the survival of thermotolerant cells was approximately 3-fold lower than expected from the mild toxicity of the drug. As this could not be accounted for by an interaction of the drug with the response of cells to single heat treatments, it is concluded that the drug inhibits the expression of thermotolerance in cells which would otherwise express a full degree of thermotolerance. The time and concentration dependence of this effect indicates that the drug acts though inhibition of rRNA synthesis. Therefore, enhanced recovery of RNA synthesis, presumably rRNA synthesis, is identified as one of the mechanisms responsible for enhanced survival of thermotolerant cells following heat shock.     

Expression, synthesis, and phosphorylation of HSP28 family during development and decay of thermotolerance in CHO plateau-phase cells.

We investigated a correlation between development of thermotolerance and expression, synthesis, or phosphorylation of HSP28 family in CHO plateau phase cells. After heating at 45.5 degrees C for 10 min, thermotolerance developed rapidly and reached its maximum 12-18 hr after heat shock. This acquired thermal resistance was maintained for 72 hr and then gradually decayed. In parallel, the levels of three 28 kDa heat shock proteins, HSP28a along with its two phosphorylated isoforms (HSP28b,c), increased and reached their maximum 24-48 hr after heat shock. The levels of these polypeptides, except HSP28c, remained elevated for 72 hr and then decreased. The level of HSP28 mRNA increased rapidly and reached its maximum 12 hr after heat shock. However, unlike thermotolerance and the levels of HSP28 family proteins, the level of HSP28 mRNA decreased rapidly within 72 hr. These results demonstrate a correlation between the amount of intracellular HSP28 family proteins and development and decay of thermotolerance.     

Cyclic AMP and the heat shock response in Chinese hamster ovary cells

Heat shock leads to transient increases in cAMP levels in HA-1-CHO cells. Such pulses are correlated temporally with the induction of heat resistance (thermotolerance) and with heat shock protein synthesis. Although the kinetics of cAMP increase after heating suggest a role in thermotolerance induction, raising cAMP levels directly using dBcAMP did not produce full thermotolerance. The resistance induced by dBcAMP may thus be either a component of or different to heat-shock triggered resistance. Cells which had been made thermotolerant by heat shock did not produce a pulse in cAMP level on heating. The cAMP producing system thus seemed desensitized to heat in thermotolerant cells.     

Small heat shock proteins and adaptation to hypertermia in various Drosophila species

Expression level and kinetics of accumulation of small heat shock proteins (21-27 kDa group) have been investigated in three Drosophila species differing significantly by temperature niche and thermosensitivity. It was shown that low-latitude thermotolerant species D. virilis exceeds the high-latitude thermosensitive closely-related species D. lummei as well as distant thermosensitive species D. melanogaster in terms of small heat shock proteins expression and accumulation after temperature elevation. The data obtained enable to postulate an important role of small heat shock proteins in organism basal thermotolerance and general adaptation to adverse conditions of environment.     

Genetic Evidence of a Role for Lck in T-Cell Receptor Function Independent or Downstream of ZAP-70/Syk Protein Tyrosine Kinases

T-cell antigen receptor (TCR) engagement results in sequential activation of the Src protein tyrosine kinases (PTKs) Lck and Fyn and the Syk PTKs, ZAP-70 and Syk. While the Src PTKs mediate the phosphorylation of TCR-associated signaling subunits and the phosphorylation and activation of the Syk PTKs, the lack of a constitutively active Syk PTK has prohibited the analysis of Lck function downstream of these initiating signaling events. We describe here the generation of an activated Syk family PTK by substituting the kinase domain of Syk for the homologous region in ZAP-70 (designated as KS for kinase swap). Expression of the KS chimera resulted in its autophosphorylation, the phosphorylation of cellular proteins, the upregulation of T-cell activation markers, and the induction of interleukin-2 gene synthesis in a TCR-independent fashion. The KS chimera and downstream ZAP-70 or Syk substrates, such as SLP-76, were still phosphorylated when expressed in Lck-deficient JCaM1.6 T cells. However, expression of the KS chimera in JCaM1.6 cells failed to rescue downstream signaling events, demonstrating a functional role for Lck beyond the activation of the ZAP-70 and Syk PTKs. These results indicate that downstream TCR signaling pathways may be differentially regulated by ZAP-70 and Lck PTKs and provide a mechanism by which effector functions may be selectively activated in response to TCR stimulation.     

Effect of heat shock on the intracellular distribution of proteins

Exposure of cells to heat induces thermotolerance, a transient resistance to subsequent heat challenges. It has been shown that thermotolerance is correlated in time with the enhanced synthesis of heat shock proteins. In this study, the association of induced heat shock proteins with various cellular fractions was investigated and the heat-induced changes in skeletal protein composition in thermotolerant and control cells was compared. All three major heat shock proteins induced in Chinese hamster fibroblasts after a 46 degrees C, 4-min heat treatment (70, 87, and 110 kDa) were purified with the cytoplasmic fraction, whereas only the 70-kDa protein was also found in other cell fractions, including that containing the cellular skeleton. Immediately after a second heat treatment at 45 degrees C for 45 min, the 110-kDa protein from thermotolerant cells also purified extensively with the cellular skeletal fraction. In this regard, the 110-kDa protein behaved similarly to many other cellular proteins, since we observed an overall temperature-dependent increase in the total labeled protein content of the high-salt-resistant cellular skeletal fraction after heat shock. Pulse-chase studies demonstrated that this increased protein content gradually returned to normal levels after a 3-hr incubation at 37 degrees C. The alteration or recovery kinetics of the total labeled protein content of the cellular skeletal fraction after heat shock did not correlate with the dramatic increase in survival observed in thermotolerant cells. The relationship between heat shock proteins and thermotolerance, therefore, does not correlate directly with changes in the heat-induced cellular alterations leading to differences in protein fractionation.     

Phosphorylation of HSP27 during development and decay of thermotolerance in Chinese hamster cells

The small molecular weight heat shock protein HSP27 was recently shown to confer a stable thermoresistant phenotype when expressed constitutively in mammalian cells after structural gene transfection. These results suggested that HSP27 may also play an important role in the development of thermotolerance, the transient ability to survive otherwise lethal heat exposure after a mild heat shock. In Chinese hamster O23 cells increased thermoresistance is first detected at 2 h after a triggering treatment of 20 min at 44 degrees C, attains a maximum at 5 hours, and decays thereafter with a half-life of 10 h. We found that the development and decay of transient thermotolerance cannot be solely explained on the basis of changes in the cellular concentration of HSP27. The cellular HSP27 concentration is not increased appreciably at 2 h after heat shock and attains a maximum at 14 h. Similar results were obtained in the case of another heat shock protein, HSP70. HSP70 follows slightly faster kinetics of accumulation (peaks at 10 h) and decays much more rapidly (ti/2 = 4h) than HSP27 (t1/2 = 13h). HSP27 has 3 isoelectric variants A, B, and C of which B and C are phosphorylated. In cells maintained at normal temperature, HSP27A represents more than 90% of all HSP27. Shifting the cell culture temperature from 37 to 44 degrees C induces the incorporation of 32P into the more acidic B and C forms, a process that occurs very rapidly since the reduction in the concentration of the A form and a corresponding increase in the level of B and C is detectable by immunoblot analysis within 2.5 min at 44 degrees C. Analyses performed at various times during development and decay of transient thermotolerance revealed a close relationship between the effect of heat shock on HSP27 phosphorylation and cell ability to survive. For example, fully thermotolerant cells (5 h post-induction) are refractory to induction of HSP27 phosphorylation by a 20-min heat shock. The induction of HSP27 phosphorylation was also studied in a family of clonal cell lines of O23 cells that are thermoresistant as a result of the constitutive expression of a transfected human HSP27 gene. In these thermoresistant cells, phosphorylation of the endogenous hamster HSP27 is induced to a level comparable to that found in the thermosensitive parental cells. However, phosphorylation of the exogenous human protein, which represents more than 80% of total HSP27 in these cells, was much less induced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermotolerance induced at a mild temperature of 40 degrees C protects cells against heat shock-induced apoptosis

Apoptosis constitutes a response of organisms to various physiological or pathological stimuli, and to different stresses. The ability of thermotolerance induced at a mild temperature of 40 degrees C to protect against activation of the apoptotic cascade by heat shock was investigated. When Chinese hamster ovary and human adenocarcinoma cervical cells were pretreated at 40 degrees C for 3 h, they were resistant to subsequent lethal heat shock at 43 degrees C. Induction of thermotolerance at 40 degrees C led to increased expression of heat shock proteins 27, 32, 72, and 90. Heat shock induced apoptotic events at the mitochondrial level, involving a decrease in membrane potential, translocation of Bax to mitochondria, and liberation of cytochrome c into the cytosol. These events were diminished in thermotolerant cells. Heat shock (42-45 degrees C) caused activation of initiator caspase-9 and effector caspases-3, -6, and -7, relative to controls at 37 degrees C. Activation of caspases was decreased in thermotolerant cells. Heat shock caused fragmentation of the caspase substrate, inhibitor of caspase-activated DNase. Fragmentation was diminished in thermotolerant cells. Thermotolerance afforded protection against heat shock-induced nuclear chromatin condensation, but not against necrosis.     

Enhanced glycosyltransferase activity during thermotolerance development in mammalian cells

The cellular heat shock response leads to the enhanced synthesis of a family of heat shock proteins and the development of thermotolerance. In CHO cells, however, heat shock also leads to enhanced synthesis of a 50 kD glycoprotein and elevated activity of N-acetylgalactosaminyltransferase (GalNAcT). In this study we showed increased GalNAcT activity during thermotolerance expression in all of five mammalian cell lines included in the study. However, there was no simple correlation between cellular heat sensitivity of unheated control cells and basal levels of GalNAcT activity, measured toward the same exogenous acceptor apomucin. Although GalNAcT was elevated in thermotolerant cells, GalNAcT activity itself did not exhibit thermotolerance in terms of reduced sensitivity to heat inactivation. The increase in GalNAcT activity after heating was similar in exponentially growing and plateau-phase cultures and was inhibited neither by cycloheximide nor actinomycin D. However, the inhibitors by themselves also increased GalNAcT activity in unheated control cells. Chemical inducers of thermotolerance (arsenite and diamide) increased GalNAcT activity, but the increase was modest when compared to that following hyperthermia. In addition to GalNAcT, two other glycosyltransferases with specificity for O-glycans, alpha 1,2-fucosyltransferase and alpha 2,6-sialyltransferase, also showed increased activity after hyperthermia and during thermotolerance development. Together with previously published data, these results support the hypothesis that heat-induced activation of O-glycan-specific glycosyltransferases plays a physiological role in the cellular heat shock response and in thermotolerance development.     

Heat shock protein synthesis and thermotolerance in Salmonella typhimurium

The resistance of stationary phase Salmonella typhimurium to heating at 55°C was greater in cells grown in nutritionally rich than in minimal media, but in all media tested resistance was enhanced by exposing cells to a primary heat shock at 48°C. Chloramphenicol reduced the acquisition of thermotolerance in all media but did not completely prevent it in any.
The onset of thermotolerance was accompanied by increased synthesis of major heat shock proteins of molecular weight about 83, 72, 64 and 25 kDa. When cells were shifted from 48°C to 37°C, however, thermotolerance was rapidly lost with no corresponding decrease in the levels of these proteins. There is thus no direct relationship between thermotolerance and the cellular content of the major heat shock proteins. One minor protein of molecular weight about 34 kDa disappeared rapidly following a temperature down-shift. Its presence in the cell was thus correlated with the thermotolerant state.     

Heat shock protein synthesis and thermotolerance in Salmonella typhimurium

The resistance of stationary phase Salmonella typhimurium to heating at 55 degrees C was greater in cells grown in nutritionally rich than in minimal media, but in all media tested resistance was enhanced by exposing cells to a primary heat shock at 48 degrees C. Chloramphenicol reduced the acquisition of thermotolerance in all media but did not completely prevent it in any. The onset of thermotolerance was accompanied by increased synthesis of major heat shock proteins of molecular weight about 83, 72, 64 and 25 kDa. When cells were shifted from 48 degrees C to 37 degrees C, however, thermotolerance was rapidly lost with no corresponding decrease in the levels of these proteins. There is thus no direct relationship between thermotolerance and the cellular content of the major heat shock proteins. One minor protein of molecular weight about 34 kDa disappeared rapidly following a temperature down-shift. Its presence in the cell was thus correlated with the thermotolerant state.     

Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells

Heat shock induces in cells the synthesis of specific proteins called heat shock proteins (HSPs) and a transient state of thermotolerance. The putative role of one of the HSPs, HSP27, as a protective molecule during thermal stress has been directly assessed by measuring the resistance to hyperthermia of Chinese hamster and mouse cells transfected with the human HSP27 gene contained in plasmid pHS2711. One- and two-dimensional gel electrophoresis of [3H]leucine- and [32P]orthophosphate-labeled proteins, coupled with immunological analysis using Ha27Ab and Hu27Ab, two rabbit antisera that specifically recognize the hamster and the human HSP27 protein respectively, were used to monitor expression and inducibility of the transfected and endogenous proteins. The human HSP27 gene cloned in pHS2711 is constitutively expressed in rodent cells, resulting in accumulation of the human HSP27 and all phosphorylated derivatives. No modification of the basal or heat-induced expression of endogenous HSPs is detected. The presence of additional HSP27 protein provides immediate protection against heat shock administered 48 h after transfection and confers a permanent thermoresistant phenotype to stable transfectant Chinese hamster and mouse cell lines. Mild heat treatment of the transfected cells results in an induction of the full complement of the endogenous heat shock proteins and a small increase in thermoresistance, but the level attained did not surpass that of heat-induced thermotolerant control cells. These results indicate that elevated levels of HSP27 is sufficient to give protection from thermal killing. It is concluded that HSP27 plays a major role in the increased thermal resistance acquired by cells after exposure to HSP inducers.     

Induction of heat shock protein 27 in rat embryos exposed to hyperthermia

Previously we reported that eight proteins were reproducibly induced in postimplantation rat embryos exposed to a brief heat shock (43°C, 15 min). The major heat-inducible rat embryo protein has now been identified as heat shock protein 72 (Hsp 72). In addition, the induction of Hsp 72 is temporally correlated with induction of thermotolerance. One of the other rat embryo proteins previously shown to be induced by elevated temperature is a heat shock protein of approximately 27 kilodaltons (Hsp 27). In this report we show that this protein is recognized by an antibody directed against a conserved peptide sequence of Hsp 27. Unlike Hsp 72, Hsp 27 is constitutively expressed in the rat embryo in the absence of any thermal stress; however, the level of Hsp 27 is increased approximately 2–3-fold after thermal stress (43°C, 10 min). Immunohistochemical analysis revealed that the constitutively expressed Hsp 27 is localized primarily to cells of the heart, cells that are uniquely resistant to the cytotoxic effects of hyperthermia. After thermal stress, Hsp 27 is expressed in all tissues of the embryo. Finally, our data show that Hsp 27 exists in the rat embryo as three major isoforms indicative of different phosphorylation states. Furthermore, most Hsp 27 in the heart is phosphorylated, whereas in the rest of the embryo, nonphosphorylated Hsp 27 predominates. After thermal stress, levels of phosphorylated isoforms increase dramatically in nonheart tissues of the embryo. Together, these results suggest that Hsp 27 may play a role in the development of thermotolerance in the postimplantation mammalian embryo. © 1996 Wiley-Liss, Inc.     

Overexpression of Akt1 upregulates glycogen synthase activity and phosphorylation of mTOR in IRS-1 knockdown HepG2 cells

Insulin receptor substrate (IRS) proteins are important docking proteins in mediating the insulin signaling cascade. We have investigated the effect of short interfering RNA (siRNA) mediated knockdown of IRS-1 on insulin signaling cascade in primary human hepatocellular carcinoma HepG2 cell line and HepG2 cells overexpressing Akt1/PKB-alpha (HepG2-CA-Akt/PKB). IRS-1 knockdown in both cell lines resulted in reduction of insulin stimulated Akt1 phosphorylation at Ser 473. In parental HepG2 cells, IRS-1 knockdown resulted in reduction (ca. 50%) in the basal level of phosphorylated mTOR (Ser 2448) irrespective of insulin treatment. In contrast, HepG2-CA-Akt/PKB cells showed an upregulation in the basal level of phosphorylated mTOR (Ser 2448) (ca. 40%). Insulin mediated phosphorylation of mTOR was reduced. IRS-1 knockdown also reduced the cell proliferation of parental HepG2 cells by ca. 30% in the presence/absence of insulin, whereas in HepG2-CA-Akt/PKB the cell proliferation was reduced by 15% and treatment of insulin further reduced it to ca. 50% (vs. control). IRS-1 knockdown also reduced the glycogen synthase (GS) activity in parental HepG2 cells, however, it was upregulated in HepG2-CA-Akt/PKB cells. These results suggest that knockdown of IRS-1 abolished basal as well as insulin mediated phosphorylation/activity of proteins involved in cell proliferation or glycogen metabolism in the parental Hep2 cells. IRS-1 knockdown in cells overexpressing constitutively active Akt1/PKB-alpha either did not change or upregulated the basal levels of phosphorylated/active proteins. However, insulin mediated response was either not altered or downregulated in these cells.