calpain and Uterine-Cervical-Neoplasms

Retinoblastoma protein (Rb) is a tumor suppressor that binds and represses E2F transcription factors. In cervical cancer cells, human papilloma virus (HPV) protein E7 binds to Rb, releasing it from E2F to promote cell cycle progression, and inducing ubiquitination of Rb. E7-mediated proteasomal degradation of Rb requires action by another protease, calpain, which cleaves Rb after Lys 810. However, it is not clear why cleavage is required for Rb degradation. Here, we report that the proteasome cannot initiate degradation efficiently on full-length Rb. Calpain cleavage exposes a region that is recognized by the proteasome, leading to rapid proteolysis of Rb. These findings identify a mechanism for regulating protein stability by controlling initiation and provide a better understanding of the molecular mechanism underlying transformation by HPV.

Topics: Acrylates; Calpain; Cell Cycle; Cell Transformation, Neoplastic; Cyclopentanes; E2F Transcription Factors; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Human papillomavirus 16; Humans; NEDD8 Protein; Oligopeptides; Papillomavirus E7 Proteins; Proteasome Endopeptidase Complex; Protein Stability; Pyrimidines; Recombinant Proteins; Retinoblastoma Binding Proteins; Ubiquitin-Protein Ligases; Ubiquitination; Uterine Cervical Neoplasms

Cyclin G2 (CCNG2) is an atypical cyclin that functions to inhibit cell cycle progression and is often dysregulated in human cancers. We have previously shown that cyclin G2 is highly unstable and can be degraded through the ubiquitin/proteasome pathway. Furthermore, cyclin G2 contains a PEST domain, which has been suggested to act as a signal for degradation by multiple proteases. In this study, we determined if calpains, a family of calcium-dependent proteases, are also involved in cyclin G2 degradation. The addition of calpain inhibitors or silencing of calpain expression by siRNAs strongly enhanced cyclin G2 levels. On the other hand, incubation of cell lysates with purified calpains or increasing the intracellular calcium concentration resulted in a decrease in cyclin G2 levels. Interestingly, the effect of calpain was found to be dependent on the phosphorylation of cyclin G2. Using a kinase inhibitor library, we found that Epidermal Growth Factor (EGF) Receptor is involved in cyclin G2 degradation and treatment with its ligand, EGF, induced cyclin G2 degradation. In addition, the presence of the PEST domain is necessary for calpain and EGF action. When the PEST domain was completely removed, calpain or EGF treatment failed to trigger degradation of cyclin G2. Taken together, these novel findings demonstrate that EGF-induced, calpain-mediated proteolysis contributes to the rapid destruction of cyclin G2 and that the PEST domain is critical for EGF/calpain actions.

Topics: Amino Acid Sequence; Animals; Calpain; Cell Line, Tumor; Cyclin G2; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Phosphorylation; Protein Domains; Proteolysis; Uterine Cervical Neoplasms

The Bcl-2 homology 3-only protein Bid is an important mediator of death receptor-induced apoptosis. Recent reports and this study suggest that Bid may also mediate genotoxic drug-induced apoptosis of various human cancer cells. Here, we characterized the role of Bid and the mechanism of Bid activation during oxaliplatin-induced apoptosis of HeLa cervical cancer cells. Small hairpin RNA-mediated silencing of Bid protected HeLa cells against both death receptor- and oxaliplatin-induced apoptosis. Expression of a Bid mutant in which caspase-8 cleavage site was mutated (D59A) reactivated oxaliplatin-induced apoptosis in Bid-deficient cells but failed to reactivate death receptor-induced apoptosis, suggesting that caspase-8-mediated Bid cleavage did not contribute to oxaliplatin-induced apoptosis. Overexpression of bcl-2 or treatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone abolished caspase-2, -8, -9, and -3 activation as well as Bid cleavage in response to oxaliplatin, suggesting that Bid cleavage occurred downstream of mitochondrial permeabilization and was predominantly mediated by caspases. We also detected an early activation of calpains in response to oxaliplatin. Calpain inhibition reduced Bid cleavage, mitochondrial depolarization, and activation of caspase-9, -3, -2, and -8 in response to oxaliplatin. Further experiments, however, suggested that Bid cleavage by calpains was not a prerequisite for oxaliplatin-induced apoptosis: single-cell imaging experiments using a yellow fluorescent protein-Bid-cyan fluorescent protein probe demonstrated translocation of full-length Bid to mitochondria that was insensitive to calpain or caspase inhibition. Moreover, calpain inhibition showed a potent protective effect in Bid-silenced cells. In conclusion, our data suggest that calpains and Bid act in a cooperative, but mutually independent, manner to mediate oxaliplatin-induced apoptosis of HeLa cells.

Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; Calpain; Female; Gene Knockdown Techniques; HCT116 Cells; HeLa Cells; Humans; Organoplatinum Compounds; Oxaliplatin; Uterine Cervical Neoplasms

We have demonstrated that soft substrate induced apoptosis in polarized cells, but not in transformed cells by disturbance of Ca(2+) homeostasis. This study aims to further investigate the regulatory mechanisms underlying the disruption of Ca(2+)-signaling integrity in soft substrate-induced epithelial apoptosis. Soft substrate up-regulated the store-operated Ca(2+) (SOC) entry across the plasma membrane of normal cervical epithelial cells, which resulted in increased cytosolic Ca(2+) levels. Concomitantly, soft substrate induced the aggregation and translocation of stromal interacting molecule 1 (STIM1) toward the cell periphery to colocalize with Orai1, an essential pore subunit of SOC channel, detected by fluorescence resonance energy transfer approach and confocal image analyses. The disturbed Ca(2+) homeostasis resulted in the activation of mu-calpain, which cleaved alpha-spectrin, induced actin disorganization, and caused apoptosis. In contrast, soft substrate did not disturb Ca(2+) homeostasis or induce apoptosis in cervical cancer cells. Chelating extracellular Ca(2+) by EGTA and down-regulated SOC entry by small interfering RNA targeting STIM1 or inhibitors targeting Ca(2+)-binding site of calpain significantly inhibited soft substrate-induced activation of mu-calpain and epithelial cell apoptosis. Thus, soft substrate up-regulates the interaction of STIM1 with SOC channels, which results in the activation of mu-calpain and subsequently induces normal epithelial cell apoptosis.

Topics: Apoptosis; Calcium; Calcium Channels; Calcium Signaling; Calpain; Cell Line; Cell Proliferation; Cervix Uteri; Collagen; Enzyme Activation; Epithelial Cells; Female; Fluorescence Resonance Energy Transfer; Homeostasis; Humans; Inositol 1,4,5-Trisphosphate; Membrane Proteins; Models, Biological; Neoplasm Proteins; ORAI1 Protein; Stromal Interaction Molecule 1; Up-Regulation; Uterine Cervical Neoplasms

Calpain 6 (Capn6) is one of the calcium-dependent intracellular nonlysosomal proteases and is known for tissue-specific expression, primarily in the placenta. We investigated whether the expression of Capn6 is associated with the progression of uterine cervical neoplasia. We analyzed 81 formalin-fixed paraffin-embedded cervical tissues that included six normal cervical epithelium, 20 low-grade squamous intraepithelial lesions (LSIL), 17 high-grade squamous intraepithelial lesions (HSIL), 20 invasive squamous cell carcinomas (ISCC) without lymph node (LN) metastasis and 18 ISCCs with LN metastasis. The expression of Capn6 was determined by immunohistochemistry and was undetectable in normal cervical squamous epithelium. The expression of the protein gradually increased in accordance with the progression from normal to LSIL (P=0.000) and from LSIL to HSIL (P=0.003). Capn6 was detected in all cases of ISCC. However, there was no significant difference between HSIL and ISCC (P=0.945), ISCC with LN metastases and ISCC without LN metastases (P=0.862). The distribution of staining was diffuse. The cytoplasm and nucleus were stained evenly. These results suggest that Capn6 may play a significant role in the development of LSIL and may also function in the progression of LSIL to HSIL. However, its role in tumor development and metastases is not clear.

Topics: Adult; Aged; Calpain; Disease Progression; Female; Humans; Immunohistochemistry; Middle Aged; Uterine Cervical Neoplasms

Different rigidities of adhesive collagen substrate affect cellular functions with unclear mechanisms. Here, we cultured a renal epithelial cell line (LLC-PK1) and a tumor cell line (HeLa) on substrates of different rigidities and compared the cell type-specific responses. The culture dish was coated with a very thin layer of collagen gel (control group) or overlaid with collagen gel (soft substrate). LLC-PK1 cells contracted as they grew on collagen gel and the apoptotic bodies obviously appeared with time. The protein levels of procaspase-12 and its downstream target procaspase-3 were decreased when LLC-PK1 cells cultured on collagen gel. Mu-calpain was activated on collagen gel. Collage gel also induced the cleavage of alpha-spectrin which resulted in the disorganization of actin cytoskeleton. In contrast, there was no significant change in cytochrome c revelation, mitochondrial membrane potential, and the protein levels of procaspase-8 and procaspase-9. Moreover, soft substrate caused elevated cytosolic Ca(2+), Ca(2+) overload in ER and upregulation of capacitative calcium entry. Ca(2+) chelator or channel blocker partially rescued the collagen-gel induced apoptosis by inhibiting mu-calpain activation. In contrast, for HeLa cells cultured either on collagen gel or on gel-coated dish, there was no significant change in positive Annexin V staining, no activation of procaspase-12 and no cleavage of mu-calpain. Thus, soft substrate induces apoptosis in LLC-PK1 cells by the disturbance of Ca(2+) homeostasis.

Topics: Actins; Animals; Apoptosis; Calcium; Calpain; Caspase 12; Caspase 3; Collagen Type I; Cytoskeleton; Endoplasmic Reticulum; Enzyme Activation; Epithelial Cells; Female; Gels; HeLa Cells; Homeostasis; Humans; Kidney; LLC-PK1 Cells; Spectrin; Swine; Time Factors; Uterine Cervical Neoplasms

Calpains are Ca(2+)-requiring, nonlysosomal proteases which are thought to participate in some aspects of intracellular Ca(2+)-signal transduction. However, their exact physiologic function has not yet been established. Addition of the cell-permeant, irreversible calpain inhibitor, ZLLY-CHN2, to human TE2 or C-33A cells inhibited growth, as assessed either by mitochondrial MTT reductase activity or by direct cell counting. Inhibition of growth produced by a 24-h exposure to 50 microM ZLLY-CHN2 was reversed upon substituting growth medium without inhibitor. Homogenates produced from cells cultured in the presence of ZLLY-CHN2 displayed decreased calpain and Ca(2+)-independent proteolytic activities. Protein immunoblot analysis showed that cell cultures which had lost 80% of their calpain activity still retained full calpain immunoreactivity. Therefore, inhibition by ZLLY-CHN2 appeared to result in accumulation of irreversibly inactivated calpain within the cells. Homogenates from cells cultured in the presence of 20 or 50 microM ZLVG-CHN2, a cell-permeant inhibitor with little activity against calpains, had decreased Ca(2+)-independent proteolytic activity, but demonstrated no decrease in calpain activity. ZLVG-CHN2 did not inhibit cell growth under these conditions. Growth of Saccharomyces cerevisiae cells, which do not appear to express calpain-like proteases, was not inhibited by including 50 microM ZLLY-CHN2 in the culture medium. These results indicate that calpains participate in the social regulation of cell growth in multicellular organisms.

Topics: Calcium-Binding Proteins; Calpain; Cell Division; Cell Line, Transformed; Cell Membrane Permeability; Cell Transformation, Viral; Cysteine Endopeptidases; Diazomethane; Female; Humans; Kinetics; Molecular Weight; Multienzyme Complexes; Oligopeptides; Protease Inhibitors; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Simian virus 40; Time Factors; Uterine Cervical Neoplasms