Usha K. Srinivas

Lack of heat shock response triggers programmed cell death in a rat histiocytic cell line

Stress response is a universal phenomenon. However, a rat histiocytic cell line, BC‐8, showed no heat shock response and failed to synthesize heat shock protein 70 (hsp70) upon heat shock at 42°C for 30 min. BC‐8 is a clone of AK‐5, a rat macrophage tumor line that is adapted to grow in culture and has the same chromosome number and tumorigenic potential as AK‐5. An increase in either the incubation temperature or time or both to BC‐8 cells leads to loss of cell viability. In addition, heat shock conditions activated apoptotic cell death in these cells as observed by cell fragmentation, formation of nuclear comets, apoptotic bodies, DNA fragmentation and activation of ICE‐like cysteine proteases. Results presented here demonstrate that BC‐8 cells cannot mount a typical heat shock response unlike all other eukaryotic cells and that in the absence of induction of hsps upon stress, these cells undergo apoptosis at 42°C.

A cross talk between cellular signalling and cellular redox state during heat-induced apoptosis in a rat histiocytoma.

Increasing evidence provides support for oxidative stress to be closely linked to apoptosis. Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. Though heat shock is a universal phenomenon, BC-8, a macrophage-like cell line failed to mount a typical heat shock response. In the absence of heat shock proteins and functional p53, BC-8 cells undergo apoptosis through CD95 signaling. In the present study, we have investigated the role of ROS in the regulation of apoptosis in these cells. We show that cells transfected with hsp70 and functional p53 are resistant to heat-induced apoptosis through inhibition of CD95 expression and ROS induction. Furthermore, apoptosis in BC-8 cells resulted in two bursts of ROS generation, one correlated with heat stress and intracellular depletion of GSH and the other with Bax overexpression and cytochrome c release. Antioxidants could not protect these cells from heat-induced apoptosis and the death pathway seems to be dependent on initial signaling cascade subsequently altering the intracellular redox. Hence, our data suggest that ROS generation in BC-8 cells upon heat shock is facultative but not obligatory for apoptosis.

Role of heat shock transcription factors in stress response and during development

A variety of physical and chemical stimuli were known to cause specific stress response in all organisms. Research done over the past 25 years has shown that there is a striking uniformity in the manner in which organisms respond to different forms of stress. Expression of stress responsive genes was shown to be regulated by two or more specific transcription factors present in the cell prior to stress. These specific genes were also activated during development. In this review, the role of heat shock transcription factors in stress response, during development and during cell cycle is described.

Cullin4B/E3-ubiquitin ligase negatively regulates β-catenin

Abstractβ-catenin is the key transducer of Wingless-type MMTV integration site family member (Wnt) signalling, upregulation of which is the cause of cancer of the colon and other tissues. In the absence of Wnt signals, β-catenin is targeted to ubiquitin-proteasome-mediated degradation. Here we present the functional characterization of E3-ubiquitin ligase encoded by cul4B. RNAi-mediated knock-down of Cul4B in a mouse cell line C3H T10 (1/2) results in an increase in β-catenin levels. Loss-of-function mutation in Drosophila cul4 also shows increased β-catenin/Armadillo levels in developing embryos and displays a characteristic naked-cuticle phenotype. Immunoprecipitation experiments suggest that Cul4B and β-catenin are part of a signal complex in Drosophila, mouse and human. These preliminary results suggest a conserved role for Cul4B in the regulation of β-catenin levels.

Heat shock transcription factors regulate heat induced cell death in a rat histiocytoma.

Heat shock response is associated with the synthesis of heat shock proteins (Hsps) which is strictly regulated by different members of heat shock transcription factors (HSFs). We previously reported that a rat histiocytoma, BC-8 failed to synthesize Hsps when subjected to typical heat shock conditions (42°C, 60 min). The lack of Hsp synthesis in these cells was due to a failure in HSF1 DNA binding activity. In the present study we report that BC-8 tumor cells when subjected to heat shock at higher temperature (43°C, 60 min) or incubation for longer time at 42°C, exhibited necrosis characteristics; however, under mild heat shock (42°C, 30 min) conditions cells showed activation of autophagy. Mild heat shock treatment induced proteolysis of HSF1, and under similar conditions we observed an increase in HSF2 expression followed by its enhanced DNA binding activity. Inhibiting HSF1 proteolysis by reversible proteasome inhibition failed to inhibit heat shock induced autophagy. Compromising HSF2 expression but not HSF1 resulted in the inhibition of autophagy, suggesting HSF2 dependent activation of autophagy. We are reporting for the first time that HSF2 is heat inducible and functions in heat shock induced autophagic cell death in BC-8 tumor cells.

Heat induced expression of CD95 and its correlation with the activation of apoptosis upon heat shock in rat histiocytic tumor cells

The heat shock response is a universal phenomenon and is among the most highly conserved cellular responses. However, BC‐8, a rat histiocytoma, fails to mount a heat shock response unlike all other eukaryotic cells. In the absence of induction of heat shock proteins, apoptotic cell death is activated in BC‐8 tumor cells upon heat shock. We demonstrate here that stable transformants of BC‐8 tumor cells transfected with hsp70 cDNA constitutively express hsp70 protein and are transiently protected from heat induced apoptosis for 6–8 h. In addition heat stress induces CD95 gene expression in these tumor cells. There is a delay in CD95 expression in hsp70 transfected cells suggesting a correlation between the cell surface expression of CD95 and the time of induction of apoptosis in this tumor cell line. Also expression of CD95 antigen appears to inhibit the interaction between heat shock factors and heat shock elements in these cells resulting in the lack of heat shock response.

Heat shock induces chromosomal instability in near-tetraploid embryonal carcinoma cells

Embryonal carcinoma cells, the cancer stem cell of teratocarcinoma, provide a good model system to study various aspects of embryonic and cancer development. Abnormal karyotype not only affects the normal development and differentiation, but also boosts tumor development. As evidences suggesting the role of karyotypic abnormalities (e.g., aneuploidy and near-tetraploidy) and stem cell in tumor development are increasing, it becomes pertinent to study karyotypically abnormal embryonal carcinoma cells. We isolated a subclone, P19RG01, from long-term culture of P19 embryonal carcinoma cell line. This subclone is near-tetraploid and expresses markers of embryonal carcinoma cells. Here we show that sub-lethal heat shock induces centrosome amplification, multipolar mitosis and chromosome missegregation thereby inducing chromosomal instability by several fold. The formation of supernumerary centrosome is accompanied by a long G2 arrest. These events lead to the generation of heterogeneous population of cells in culture. Our observations support the previous observations that selective forces (both intrinsic as well as extrinsic) play an important role in tumor progression. Moreover, we provide direct evidences which suggest chromosomal instability in cancer stem cells, using P19RG01 embryonal carcinoma cells as a model system. We propose that a minor population of chromosomally unstable cancer stem cells exists in a tumor or is generated (transient existence) in the presence of selective forces. These chromosomally unstable cancer stem cells generate a heterogeneous cancer stem cell population, which undergoes Darwinian selection, leading to selection of more malignant cancer stem cells and thereby tumor progression.

Activation of stress response by ionomycin in rat hepatoma cells

All living systems respond to a variety of stress conditions by inducing the synthesis of stress or heat shock proteins (HSPs), which transiently protect cells. HSP synthesis was preceded by an increase in intracellular free calcium concentration [(Ca2+)i]. In this study, we show that Ca2+ ionophore, ionomycin, induced an immediate increase in intracellular free Ca2+ and examined how this increase affects heat shock response in rat hepatoma cell line H4II‐E‐C3. Results indicate that incubating H4II‐E‐C3 cells with 0.3 μM ionomycin at 37°C for 15 min results in the induction of HSP 70 in both Ca2+‐containing and Ca2+‐free medium. Associated with this increase in free Ca2+ is an in vivo change in membrane organization and activation of signaling molecules like ERKS and SAPKs/JNK. In Ca2+ containing medium HSP 70 induction mediated by HSF–HSE interaction was faster upon ionomycin treatment as compared to heat shock. Our results show that ionomycin, at sub lethal concentration, increases intracellular free Ca2+ concentration, activates SAPK/JNK and HSF–HSE interaction, and induces HSP 70 synthesis. J. Cell. Biochem. 86: 154–161, 2002.

Heat-induced expression of albumin during early stages of rat embryo development.

An examination of heat-induced expression of proteins in tissues from adult and embryonic liver in rats shows that albumin, which is constitutively expressed in adult liver and is not synthesized in embryos before 16 days of gestation, appears in liver cells at earlier stages of development upon heat shock. On the basis of available evidence for the expression of heat shock proteins at distinct stages of development and on the basis of our findings, it may be argued that there could be common molecular events taking place during development and as a result of heat shock. We suggest also that one of the consequences of heat shock could be an internal change of pH within the cell which, in turn, might trigger alterations in gene expression.

Cloning and characterization of mouse cullin4B/E3 ubiquitin ligase.

Heat induced differentiation of mouse embryonal carcinoma cells PCC4 has been reported earlier. We have further characterized the phenotype of the differentiated cells and by DD-RT-PCR identified several partial cDNAs that are differentially expressed during differentiation. Nucleotide homology search revealed that the genes corresponding to some of the up-regulated partial cDNAs are indeed part of differentiation pathway. 5′ extension of an EST that has homology to one of the partial cDNAs led to the identification of mouse cullin4B. Cullin4B is coded by a separate gene and has a unique and longer amino-terminal end with a putative nuclear localization signal sequence (NLS). We have cloned, expressed and raised antibodies against the amino and carboxy-terminal halves of cullin4B. Immuno staining of differentiated PCC4 cells with N-terminal Cul4B antibody showed enhanced expression of Cul4B and its translocation into the nucleus upon differentiation. Transient transfection of a chimeric gene encoding the N-terminal part of Cul4B fused to green fluorescent protein into PCC4 cells revealed that the protein was localized in the nucleus confirming the functional significance of the putative NLS. Since cullins are involved in recognition of specific proteins for degradation, based on the evidence presented here, we hypothesize that cullin4B is probably involved in differentiation specific degradation/ modification of nuclear proteins.