N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide promotes bone formation via the canonical Wnt/ß-catenin signaling pathway.

Osteoporosis is characterized by low bone mineral density and microarchitectural deterioration of bone tissue. N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (MBOC) is one of the macamides isolated from Maca (Lepidium meyenii Walp.), a cruciferous plant from the Andes of Peru. In this study, C3H/10T1/2 mesenchymal stem cells were treated with MBOC in osteogenic induction medium. An ovariectomized (OVX) mouse model was used to investigate the effect of 1-month MBOC treatment on the prevention of postmenopausal osteoporosis. Remarkably, trabecular thickness, trabecular number, and bone volume/tissue volume of the distal femoral metaphysis were significantly increased in OVX + MBOC mice compared with OVX mice, as revealed by microcomputed tomography analysis. Trabecular separation was decreased in OVX + MBOC mice compared with OVX mice. Consistently, MBOC increased the levels of osteocalcin and runt-related transcription factor 2 in OVX mice, as well as the expression of runt-related transcription factor 2, osterix, and alkaline phosphatase in C3H/10T1/2 cells. Mechanistically, MBOC activates the canonical Wnt/ß-catenin signaling pathway via inhibiting phosphorylation of GSK-3ß at Tyr216 and maintaining ß-catenin expression. Collectively, the current study demonstrates the robustness of MBOC in the induction of mesenchymal stem cells osteogenic differentiation and consequent bone formation, suggesting that MBOC may be a potentially effective drug to treat postmenopausal osteoporosis.

Neuronal mTORC1 Is Required for Maintaining the Nonreactive State of Astrocytes.

Astrocytes respond to CNS insults through reactive astrogliosis, but the underlying mechanisms are unclear. In this study, we show that inactivation of mechanistic target of rapamycin complex (mTORC1) signaling in postnatal neurons induces reactive astrogliosis in mice. Ablation of Raptor (an mTORC1-specific component) in postmitotic neurons abolished mTORC1 activity and produced neurons with smaller soma and fewer dendrites, resulting in microcephaly and aberrant behavior in adult mice. Interestingly, extensive astrogliosis without significant astrocyte proliferation and glial scar formation was observed in these mice. The inhibition of neuronal mTORC1 may activate astrogliosis by reducing neuron-derived fibroblast growth factor 2 (FGF-2), which might trigger FGF receptor signaling in astrocytes to maintain their nonreactive state, and FGF-2 injection successfully prevented astrogliosis in Raptor knock-out mice. This study demonstrates that neuronal mTORC1 inhibits reactive astrogliosis and plays an important role in CNS pathologies.

Synergistic effects of atorvastatin and rosiglitazone on endothelium protection in rats with dyslipidemia.

BACKGROUND: Endothelial dysfunction is implicated in the initiation and progression of atherosclerosis. Whether atorvastatin combined with rosiglitazone has synergistic effects on endothelial function improvement in the setting of dyslipidemia is unknown. METHODS: Dyslipidemia rat model was produced with high-fat and high-cholesterol diet administration. Thereafter, atorvastatin, rosiglitazone or atorvastatin combined with rosiglitazone were prescribed for 2 weeks. At baseline, 6 weeks of dyslipidemia model production, and 2 weeks of medical intervention, fasting blood was drawn for parameters of interest evaluation. At the end, myocardium was used for 15-deoxy-delta-12,14-PGJ2 (15-d-PGJ2) assessment. RESULTS: Initially, there was no significant difference of parameters between sham and dyslipidemia groups. With 6 weeks' high-fat and high-cholesterol diet administration, as compared to sham group, serum levels of triglyceride (TG), total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C) were significantly increased. Additionally, nitric oxide (NO) production was reduced and serum levels of malondialdehyde (MDA), C-reactive protein (CRP) and asymmetric dimethylarginine (ADMA) were profoundly elevated in dyslipidemia group. After 2 weeks' medical intervention, lipid profile was slightly improved in atorvastatin and combined groups as compared to control group. Nevertheless, in comparison to control group, NO production was profoundly increased and serum levels of MDA, CRP and ADMA were significantly decreased with atorvastatin or rosiglitazone therapy. 15-d-PGJ2 expression of myocardium was also significantly elevated with atorvastatin or rosiglitazone treatment. Notably, these effects were further enhanced with combined therapy, suggesting that atorvastatin and rosiglitazone had synergistic effects on endothelial protection, and inflammation and oxidation amelioration. CONCLUSION: Atorvastatin and rosiglitazone therapy had synergistic effects on endothelium protection as well as amelioration of oxidative stress and inflammatory reaction in rats with dyslipidemia.

[Effect of metformin on apoptosis of renal cell carcinoma cells in vitro and its mechanisms].

OBJECTIVE: To evaluate the effect of metformin on the apoptosis of renal cell carcinoma (RCC) cells in vitro and its mechanisms. METHODS: Fluorescent microscopy and flow cytometry were used to examine the changes in the apoptosis of 786-O cells after metformin treatment. The possible signaling molecules involved in this process were analyzed by immunoblot analysis of AMP-activated protein kinase (AMPK) signaling and caspase 9. RESULTS: Metformin induced apoptosis and caspase 9 activation in 786-O cells in low-serum medium but not in normal-serum medium. Metformin also induced AMPK activation in 786-O cells, but this activation was not associated with the cell proliferation inhibition or apoptosis-inducing effect of metformin. CONCLUSION: Metformin can induce apoptosis of RCC cells in vitro, suggesting its potential as a therapeutic agent for RCC.

[Protein kinase D3 is involved in negative regulation of MMP-7 in prostate cancer cells].

OBJECTIVE: To explore the role of protein kinase D3 (PKD3) in the regulation of matrix metalloproteinases 7 (MMP-7) expression in prostate cancer cells. METHODS: PC-3 cells were either stimulated with 100 nmol/L PMA to activate PKD3 kinase activity, or transiently transfected with PKD3 siRNA, and the relative expression level of MMP-7 mRNA were analyzed by real-time PCR using 2(-delta delta Ct) method. MMP-7 mRNA levels were also analyzed and quantified in HEK293 cells with over-expression of wild-type PKD3, PKD3 knockdown (using PKD3 siRNA), or over-expression of wild-type PKD3 followed by PKD3 knockdown. RESULTS: MMP-7 mRNA expression in PC3 cells was significantly decreased after PMA-induced PKD3 kinase activation. In contrast, PKD3 knockdown by siRNA transfection markedly increased MMP-7 mRNA level (P<0.01). MMP-7 mRNA level in HEK293 cells was significantly decreased by PKD3 over-expression, whereas obviously increased by PKD3 knockdown. Down-regulation of MMP-7 mRNA level in HEK293 induced by PKD3 over-expression was rescued by PKD3 knockdown. CONCLUSION: PKD3 may contribute to the malignant progression of prostate cancer cells through negative regulation of MMP-7 expression.

[PKD3 contributes to up-regulation of prostate-specific antigen in prostate cancer cells].

OBJECTIVE: To investigate the role of PKD3 in prostate-specific antigen (PSA) expression regulation in androgen-dependent prostate cancer cells and explore the mechanism. METHODS: LNCaP cells containing low level of PKD3 were transfected with pEGFP-C2 or pEGFP-PKD3 plasmid followed by dihydrotestosterone (DHT) treatment, and PSA mRNA level was analyzed by RT-QPCR using 2(-delta delta Ct) method. Wild-type or kinase-dead PKD3 plasmids, human androgen receptor plasmid pSVAR0, pMMTV-luc of AR luciferase reporter and renilla luciferase reporter pRL-SV40 were cotransfected into HEK293 cells, and after treatment with DHT for 24 h, the cells were harvested and AR transcriptional activity were determined by dual-luciferase reporter assay. The subcellular localization of endogenous PKD3 and AR and their colocalization induced by DHT were observed by confocal microscopy. RESULTS: PSA mRNA level triggered by DHT was significantly increased by overexpression of pEGFP-PKD3 in LNCaP cells compared with that in pEGFP-C2 control cells (P<0.001). AR transcription in response to DHT treatment was also significantly up-regulated by wild type PKD3 expression (P<0.001), but partially down-regulated by kinase-dead PKD3 mutant (P<0.01). Endogenous PKD3 and AR in LNCaP cells not only translocated from the cytoplasm to the nucleus, but also colocalized with each other after DHT stimulation. CONCLUSION: Elevated AR transcriptional activity and enhanced expression of PSA induced by PKD3 in response to DHT treatment suggest that PKD3 contributes to the proliferation and malignant growth of androgen-dependent prostate cancer cells.

[Effects of blocking phospholipase C-gamma1 signaling pathway on proliferation and apoptosis of human colorectal cancer cell line LoVo].

BACKGROUND & OBJECTIVE: Phospholipase C-gamma 1 (PLC-gamma1) is a vital signal transducer in transmembrane signaling, which regulates cell proliferation and apoptosis. It is overexpressed in many cancers, such as colorectal cancer, which indicates that it is closely related to the genesis and development of tumors. This study was to explore the effects of blocking PLC-gamma1 signaling pathway on the proliferation and apoptosis of human colorectal cancer cell line LoVo, and investigate the signaling mechanisms. METHODS: LoVo cells were treated with PLC-gamma1-specific chemical blocking agent U73122. Cell proliferation was examined by cell counting, MTT assay, and flow cytometry (FCM). Cell apoptosis was observed under a microscope, and measured by agarose gel electrophoresis and FCM with PI simple staining. The expression of hot shock protein 70(HSP70) and Caspase-3 in LoVo cells were detected by Western blot. RESULTS: The proliferation of LoVo cells was inhibited after blocking PLC-gamma1 signaling pathway and the effect was enhanced along with the increasing concentration of U73122. The inhibition rate reached 35% and 45% when treated with 10 micromol/L U73122 for 24 h and 48 h respectively. After blocking PLC-gamma1 signaling pathway, the G1 phase proportion of LoVo cells was increased while the S phase proportion was decreasedû no apoptosis-specific cell shrinkage was found under a light microscope, and no apoptosis-specific DNA ladder was found by agarose gel electrophoresisû no activated Caspase-3 was detected by Western blot, while increased expression of HSP70 was detected. CONCLUSIONS: Blocking PLC-gamma1 signaling pathway can inhibit the proliferation and cell cycle progress of LoVo cells, which may be due to the up-regulated expression of HSP70. PLC-gamma1 is not a vital signal molecule regulating the apoptosis of LoVo cells.

[Phosphoprotein affinity profiling--a novel method for screening regulators of lipopolysaccharide signaling pathway].

OBJECTIVE: To To establish a method of phosphoprotein affinity profiling for identifying phosphoproteomic differences between Thp-1 cells with or without lipopolysaccharide (LPS) stimulation, aiming to screen potential regulators involved in LPS pathway. METHODS: Thp-1 cells were stimulated with 100 ng/ml PMA for 48 h to induce differentiation into mature macrophages, which, after culture for another 48 h in the absence of PMA, were either treated with 100 ng/ml LPS for 30 min or left untreated. After desalting procedure with ultrafiltration, the phosphoproteins enriched by phosphoprotein metal affinity column (PMAC) of both groups were run on 2-D electrophoresis to find the spots with different phosphorylation status. Finally, some of these spots were identified by mass spectrometry (MS) and subsequent bioinformatic analysis. RESULTS: Compared to untreated Thp-1 cells, LPS stimulated Thp-1 cells showed 29 spots with reproducible alterations on the 2-D map, including 8 representing up-regulated spots, 7 new spots, 10 down-regulated spots, and 4 absent spots. The newly emerged and absent protein spots were subjected to MS analysis, and 4 of them were identified to be involved in various cellular processes such as proteolysis, signal transduction and protein folding. Among these, phosphorylation of proteasome C2 subunit was dramatically up-regulated in LPS-stimulated cells, as was consistent with previous reports; the phosphorylation of Z-DNA-binding protein 1 has not been reported so far and needs further confirmation. CONCLUSION: Phosphoprotein affinity profiling is an attractive method for screening novel regulators involved in LPS signaling pathways and can be widely used in systemic study of signal transduction.

Antitumour effects on human colorectal carcinomas cells by stable silencing of phospholipase C-gamma 1 with lentivirus-delivered siRNA.

BACKGROUND: In most colorectal carcinomas, the level of phospholipase C (PLC)-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells. METHODS: Recombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed. RESULTS: Stable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined. Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis (30%-40%) of LoVo cells. CONCLUSIONS: PLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas.

[Effects of pentobarbital sodium on rhythmical respiration of neonatal rat medullary preparations].

OBJECTIVE: To study the effects of pentobarbital sodium in generation and modulation of rhythmical respiration in neonatal rats. METHODS: The effects of pentobarbital sodium were examined on hypoglossal nerve (XII) rootlets and inspiratory neurons in the medullary preparations including the medial region of the nucleus retrofacialis, pre-Bötzinger complex and the dorsal respiratory group of neonatal rats aged 0-3 days. The electrical activity of XII nerve rootlets and inspiratory neurons were recorded. Different doses of pentobarbital sodium (20, 40, 60, 80 micromol/L) were added into modified Krebs solution to observe changes in the discharge activity of XII nerve and inspiratory neurons. Bicuculline was used to further investigate the mechanisms that pentobarbital sodium suppresses respiration. RESULTS: The discharge activity inhibition of XII nerve was increased as pentobarbital sodium doses increased from 20 to 60 micromol/L, but no significant difference was observed between the doses of 60 and 80 micromol/L. Bicuculline can partly restore the rhythmical respiration discharge activity. CONCLUSION: Pentobarbital sodium can suppress respiration partly via GABAA receptors.

Changes of the proportion and mortality of pulmonary thromboembolism in hospitalized patients from 1974 to 2005.

BACKGROUND: Pulmonary thromboembolism (PTE) has become a common disease that severely endangers people's health. This study analysed the changes in proportion and mortality of PTE in hospitalized patients to provide data for prevention and management of the disease. METHODS: The data of 763 hospitalized patients with PTE from 1974 to 2005 in Fuwai Hospital were analysed. RESULTS: During the 1970s, 0.27% of patients in a cardiovascular hospital had PTE (< 5 cases per year); while so far this century the proportion is 0.94% (48 to 113 per year). The mortality of hospitalized PTE patients fell from 20.00% in the 1970s to 4.10% this century. Prior to 1990, the mortality of hospitalized PTE patients was 12.50%, and in the years after 1990 only 3.40%. The difference was statistically significant (P < 0.005). People with this disease were mostly between the ages of 30 and 69 years. Men were most susceptible between the ages of 30 and 69 years, while women between the ages of 40 and 69 years. Men contracted PTE 10 years earlier than women. The mortality of male PTE patients was 4.70%, not significantly different from female patients, 5.06% (0.50 < P < 0.75). There were not any significant differences between the mortality of patients in the different age groups overall (< or = 39, 40 - 49, 50 - 59, and > or = 60 years, P > 0.1). More people contracted the disease in winter than in other seasons (P < 0.05). There was no obvious difference between the mortality in different seasons overall (0.75 < P < 0.90). CONCLUSION: PTE is an increasingly significant disease and deserves adequate attention.

[Vicissitudes of proportion and mortality of patients with pulmonary thromboembolism].

OBJECTIVE: To investigate the vicissitudes of the proportion and the mortality of patients with pulmonary thromboembolism (PTE). METHODS: To analyze respectively the data of 442 patients with PTE admitted to the Fuwai Hospital during the period 1974-2002 were analyzed retrospectively. RESULTS: The numbers of PTE in-patients every year during the 1970s and 48-79 cases in the early 2000s. The hospitalization constituent ratio of PTE was 0.1152% in 1970s and was 0.5866% in early 2000s. The age of onset ranged from 30 to 60. The tide age was from 30 to 60 in the male patients and from 40 to 60 in the female patients. The in-hospital mortality of PTE patients was descending from 20.0% in 1970s to 5.8% in early 2000s. The in-hospital mortality was 12.5% before 1990 and was 4.5% after 1990 (P = 0. 005). The in-hospital mortality rates of the male and female patients were 4.6% and 8.6% respectively (P = 0.09). The in-hospital mortality rates of the patients younger than 70 and elder than 70 were 5.5% and 19.0% respectively (P < 0.05). CONCLUSION: PTE The mortality of PTE, which attacks males more frequently than females, and the proportions of the number of PTE in-patients to the number of the whole in-patients increased and the in-hospital mortality increased during the past 3 decades.

Reactive oxygen species stimulates receptor activator of NF-kappaB ligand expression in osteoblast.

It has been established that reactive oxygen species (ROS) such as H2O2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-kappaB ligand (RANKL) is a critical osteoclastogenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells.

[Effects of underlying cerebrocardiovascular diseases on the incidence of critical conditions and multiple organs dysfunction syndrome in severe acute respiratory syndrome cases].

OBJECTIVE: To investigate the effects of underlying cerebrocardiovascular diseases on the incidence of critical conditions and multiple organs dysfunction syndrome (MODS) among the severe acute respiratory syndrome (SARS) cases. METHODS: A database of all SARS cases in Beijing in 2003 was established and the data of 1291 cases whose data were complete among them was analyzed. The effects of cerebrocardiovascular diseases, other comorbid diseases and absence of underlying diseases on the incidence of critical conditions and MODS in SARS cases were compared with chi-square test and logistic regression analysis. RESULTS: The incidence rates of critical conditions and MODS in SARS cases were 34.86% and 13.71% respectively. The incidence rates of critical conditions and MODS among the SARS cases without underlying diseases, with other comorbid diseases, and with cerebrocardiovascular diseases were 28.10%, 43.70%, and 58.18%, and 9.83%, 18.52%, and 27.27% respectively. The differences among the three groups were all statistically significant (all P < 0.0001). Logistic regression analysis showed that presence of cerebrocardiovascular diseases in SARS patients was independently associated with the risks of critical conditions and MODS in comparison with those without underlying diseases after adjusting for age and occupation. [odds ratio (OR) = 1.819, 95% CI = 1.276 approximately 2.592, and OR = 1.974, 95% CI = 1.273 approximately 3.060 respectively]. CONCLUSION: The SARS cases with cerebrocardiovascular diseases have the highest incidence rate of critical conditions and MODS. Presence of Cerebrocardiovascular diseases is an important risk factor for critical conditions and MODS in the SARS cases.

Antisense epidermal growth factor receptor (EGFR) transfection down-regulates EGFR expression and suppresses the malignant phenotype of human nasopharyngeal carcinoma CNE-2 cell line.

OBJECTIVE: To determine whether epidermal growth factor receptor (EGFR) expression contributes to tumor growth of poorly differentiated human nasopharyngeal carcinoma CNE-2 cell lines. METHODS: An expression vector containing a N-terminal fragment (1.35 kb) of human EGFR in the antisense orientation was transfected into CNE-2 cell lines via lipofectamine. The established clones resistant to G418 were isolated and characterized, and the tumor-inhibiting effect of antisense EGFR expression was evaluated in terms of tumor growth and metastasis at different time after subcutaneous inoculation into nude mice. RESULTS: Down-regulated EGFR expression in the cells with antisense vector transfection was demonstrated by ligand binding assay. The growth rate and the ability to grow in soft agarose of these antisense transfectants were also reduced. After inoculation into nude mice, EGFR antisense transfectants showed a longer latency period, slower tumor growth and lower metastatic rates to the lymph nodes and lung in comparison with the parental cells. CONCLUSIONS: These results suggest that these EGFR antisense cDNA-transfected CNE-2 cells are of value to further delineate the role of EGFR in the development and progression of nasopharyngeal carcinoma.

Phospholipase C-gamma1 is required for cell survival in oxidative stress by protein kinase C.

Phospholipase C-gamma1 (PLC-gamma1) activation has been reported to enhance cell survival during the cellular response to oxidative stress. We studied the role of protein kinase C (PKC) pathways in mediating PLC-gamma1 survival signalling in oxidative stress by using mouse embryonic fibroblasts genetically deficient in PLC-gamma1 (Plcg1(-/-)) and its wild type (Plcg1(+/+)). PLC-gamma1 was activated by H(2)O(2) treatment in a dose- and time-dependent manner. Activation of PKC was also markedly increased in both cell lines treated with H(2)O(2) (1-5 mM), but with low doses (50-200 microM), PKC activation was considerably decreased in Plcg1(-/-) cells. After treatment with H(2)O(2), PKC-dependent phosphorylation of Bcl-2 and cell viability of Plcg1(-/-) cells decreased dramatically and caspase-3-like activity increased significantly compared with that of the wild-type cells. Furthermore, pretreatment of Plcg1(+/+) cells with PKC-specific inhibitor decreased levels of PKC-dependent Bcl-2 phosphorylation, enhanced caspase-3 activity and their sensitivity to H(2)O(2). On the contrary, treatment of Plcg1(-/-) cells with PKC-specific activator increased the Bcl-2 phosphorylation, decreased caspase-3 activity and improved their survival. These results suggest that PLC-gamma1 mediates survival signalling in oxidative-stress response by PKC-dependent phosphorylation of Bcl-2 and inhibition of caspase-3.

Phospholipase C-gamma1 is required for survival in heat stress: involvement of protein kinase C-dependent Bcl-2 phosphorylation.

The consequences of heat-induced phospholipase C-gamma1 (PLC-gamma1) phosphorylation are not known. We investigated the role of PLC-gamma1 activation and its downstream targets during the cellular response to heat stress using mouse embryonic fibroblasts genetically deficient in PLC-gamma1 (Plcg1 null MEF) and its wild type (wt MEF) as models. Treatment of wt MEF with heat resulted in temperature- and heating duration-dependent tyrosine phosphorylation of PLC-gamma1. HSP70 synthesis and the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) and c-Jun N-terminal protein kinase (JNK) increased equally following heat treatment in both cell lines. However, heat-induced protein kinase C (PKC) activation was dramatically reduced in Plcg1 null MEF compared with wt MEF. Importantly, the mitochondrial localization of PKCalpha, PKC-dependent phosphorylation of Bcl-2, and cell viability in Plcg1 null MEF following heat treatment, were significantly decreased compared with the wild type. Furthermore, pretreatment with bryostatin-1, a PKC activator, enhanced Bcl-2 phosphorylation and cellular resistance to heat-induced apoptosis in Plcg1 null MEF. Taken together, these results suggest that PLC-gamma1 activation enhances cell survival through the PKC-dependent phosphorylation of Bcl-2 during the cellular response to heat stress.