calpain and Cell-Transformation--Neoplastic

Calpains represent a well conserved family of calcium-dependent proteolytic enzymes. Recent progress in determining the three-dimensional crystal structure of calpains and generation of calpain knock out animals have significantly advanced our understanding of both the activation mechanism and physiological role of this protease family. Studies applying molecular intervention strategies and genetic ablation of calpain now provide indisputable evidence that calpain activity contributes to remodelling of the actin cytoskeleton, cell migration and oncogenic transformation. Src and epidermal growth factor receptor (EGFR) stimulated cell motility is dependent upon calpain activation. In addition, calpain promotes accelerated cell-cycle progression and anchorage-independent growth of Src transformed cells. In vivo studies demonstrate a link between calpain expression levels and activity with tumour development and invasion. Thus, recent investigations suggest that the role of calpain in promoting cell transformation and cell migration may have important in vivo consequences in the context of cancer pathobiology.

Topics: Animals; Calpain; Cell Movement; Cell Transformation, Neoplastic; Humans; Neoplasms; Protein Conformation

Lung cancer is diagnosed at an advanced stage due to its unrecognized symptoms, resulting in high mortality. In recent decades, research into the development of an early diagnostic method for lung cancer has expanded in order to overcome the high mortality rate. Calpain 2 (CAPN2) has been suggested as a tumor marker linked to angiogenesis, cell proliferation, and migration in non-small cell lung cancer. In this study, CAPN2 enzyme-activatable near-infrared peptide sensor linked to human serum albumin (HSA-CAPN2) was developed. Intracellular localization and strong recovered fluorescence signals of HSA-CAPN2 were observed in

Topics: A549 Cells; Animals; Biomarkers, Tumor; Biosensing Techniques; Calpain; Carcinoma, Non-Small-Cell Lung; Cell Transformation, Neoplastic; Early Detection of Cancer; Humans; Lung Neoplasms; Mice; Nanotechnology; Optical Imaging; Serum Albumin, Human

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

Topics: Animals; Calpain; Cell Transformation, Neoplastic; GATA1 Transcription Factor; Humans; Leukemia; Megakaryocytes; Mutation; Positive Transcriptional Elongation Factor B; Ribonucleoproteins, Small Nuclear

Megakaryocyte morphogenesis employs a "hypertrophy-like" developmental program that is dependent on P-TEFb kinase activation and cytoskeletal remodeling. P-TEFb activation classically occurs by a feedback-regulated process of signal-induced, reversible release of active Cdk9-cyclin T modules from large, inactive 7SK small nuclear ribonucleoprotein particle (snRNP) complexes. Here, we have identified an alternative pathway of irreversible P-TEFb activation in megakaryopoiesis that is mediated by dissolution of the 7SK snRNP complex. In this pathway, calpain 2 cleavage of the core 7SK snRNP component MePCE promoted P-TEFb release and consequent upregulation of a cohort of cytoskeleton remodeling factors, including α-actinin-1. In a subset of human megakaryocytic leukemias, the transcription factor GATA1 undergoes truncating mutation (GATA1s). Here, we linked the GATA1s mutation to defects in megakaryocytic upregulation of calpain 2 and of P-TEFb-dependent cytoskeletal remodeling factors. Restoring calpain 2 expression in GATA1s mutant megakaryocytes rescued normal development, implicating this morphogenetic pathway as a target in human leukemogenesis.

Topics: Actinin; Animals; Blotting, Western; Calpain; Cell Differentiation; Cell Nucleus; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Cytoskeleton; Flow Cytometry; GATA1 Transcription Factor; Humans; Immunoprecipitation; Leukemia; Megakaryocytes; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Morphogenesis; Mutation; Positive Transcriptional Elongation Factor B; Protein Binding; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleoproteins, Small Nuclear; RNA, Messenger; Transcription, Genetic

Ground glass hepatocytes harboring hepatitis B virus (HBV) pre-S2 mutants have been recognized as pre-neoplastic lesions of hepatocellular carcinoma (HCC). The pre-S2 mutants accumulated in endoplasmic reticulum (ER) can induce ER stress, upregulate cyclin A and promote hepatocyte proliferation. Notably, cyclin A was aberrantly detected in the cytoplasm, instead of nucleus, of pre-S2 mutant-transgenic mice livers, thereby raising the potential role of cytoplasmic cyclin A in HBV hepatocarcinogenesis. In this study, we confirmed that cyclin A was detected in the cytoplasm in the majority of HBV-related HCC tissues. In vitro, the pre-S2 mutant-initiated ER stress could induce cytoplasmic cyclin A mediated via cleavage by the calcium-dependent protease μ-calpain, resulting in an N-terminal truncated product which was preferentially located in the cytoplasm. The aberrant cyclin A expression subsequently induced centrosome overduplication, and this effect was abolished by calpain-specific inhibitors or RNA interference targeting to cyclin A. Overall, our data indicate that HBV pre-S2 mutant may elicit aberrant cyclin A expression and centrosome overduplication through ER stress induction and thereby represent a potential mechanism for the chromosome instability in HBV hepatocarcinogenesis.

Topics: Animals; Calpain; Carcinoma, Hepatocellular; Cell Growth Processes; Cell Transformation, Neoplastic; Centrosome; CHO Cells; Chromosomal Instability; Cricetinae; Cyclin A; Endoplasmic Reticulum; Hepatitis B Surface Antigens; Hepatitis B virus; Hepatocytes; Humans; Liver; Liver Neoplasms; Mice; Mice, Transgenic; Mutation; Protein Precursors; RNA Interference; Tumor Cells, Cultured

The lymphovascular embolus is an enigmatic entity adept at metastatic dissemination and chemotherapy resistance. Using MARY-X, a human breast cancer xenograft that exhibits florid lymphovascular emboli in mice and spheroids in vitro, we established a model where the in vitro transition stages from minced tumoral aggregates to well-formed spheroids served as a surrogate for in vivo emboli formation. MARY-X well-formed spheroids and emboli exhibited strong similarity of expression. The aggregate-to-spheroid transition stages were characterized by increased ExoC5, decreased Hgs and Rab7, increased calpains, increased full-length E-cadherin (E-cad/FL), and the transient appearance of E-cad/NTF2, a 95 kDa E-cadherin fragment and increased Notch3icd (N3icd), the latter two fragments produced by increased γ-secretase. Both transient and permanent knockdowns of Rab7 in MCF-7 cells increased protein but not transcription of E-cad/FL and resulted in the de novo appearance of E-cad/NTF2, the presence of nuclear E-cad/CTF2, and increased Notch1icd (N1icd). Overexpression of Rab7 conversely decreased E-cad/FL, γ-secretase (PS1/NTF), and E-cad/NTF2. Overexpression of calpains did not alter PS1/NTF but decreased E-cad/FL and E-cad/NTF2 and increased N1icd. Well-formed spheroids showed increased Rab7, absent E-cad/NTF2, decreased PS1/NTF, increased E-cad/NTF1, and increased N3icd, the latter two fragments being the direct and indirect consequences, respectively, of increased calpains (calpain 1 and calpain 2). Inhibition of calpains decreased E-cad/NTF1 but increased E-cad/NTF2 showing that calpains compete with γ-secretase (PS1) for closely located cleavage/binding sites on E-cadherin and that increased calpains can shuttle even decreased levels of γ-secretase to Notch 3, resulting in increased Notch 3 signaling in the well-formed spheroids.

Topics: Amyloid Precursor Protein Secretases; Animals; Blotting, Western; Breast Neoplasms; Cadherins; Calpain; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Endosomal Sorting Complexes Required for Transport; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, SCID; Neoplastic Cells, Circulating; Oligonucleotide Array Sequence Analysis; Phosphoproteins; Proteolysis; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins; Receptor, Notch3; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Spheroids, Cellular; Transplantation, Heterologous

Since calpain 6 is overexpressed in uterine cervical cancer tissue compared to normal tissue, we sought to define the role of calpain 6 during tumorigenesis. We overexpressed calpain 6 or inhibited calpain 6 in human cervical cancer cells (HeLa cells) and human umbilical vein endothelial cells (HUVECs), and measured cisplatin-mediated apoptosis and VEGF-mediated angiogenesis. The results indicated that calpain 6 supported tumorigenesis by inhibiting apoptosis and facilitating angiogenesis. To our knowledge, this result is the first evidence implicating calpain 6 in tumorigenesis, and it reveals calpain 6 as a novel therapeutic target for certain types of cancers.

Topics: Antineoplastic Agents; Apoptosis; Base Sequence; Blotting, Western; Calpain; Cell Transformation, Neoplastic; Cisplatin; Cloning, Molecular; DNA Primers; DNA, Complementary; Female; HeLa Cells; Humans; Neovascularization, Pathologic; Reverse Transcriptase Polymerase Chain Reaction

v-Src-induced oncogenic transformation is characterized by alterations in cell morphology, adhesion, motility, survival, and proliferation. To further elucidate some of the signaling pathways downstream of v-Src that are responsible for the transformed cell phenotype, we have investigated the role that the calpain-calpastatin proteolytic system plays during oncogenic transformation induced by v-Src. We recently reported that v-Src-induced transformation of chicken embryo fibroblasts is accompanied by calpain-mediated proteolytic cleavage of the focal adhesion kinase (FAK) and disassembly of the focal adhesion complex. In this study we have characterized a positive feedback loop whereby activation of v-Src increases protein synthesis of calpain II, resulting in degradation of its endogenous inhibitor calpastatin. Reconstitution of calpastatin levels by overexpression of exogenous calpastatin suppresses proteolytic cleavage of FAK, morphological transformation, and anchorage-independent growth. Furthermore, calpastatin overexpression represses progression of v-Src-transformed cells through the G(1) stage of the cell cycle, which correlates with decreased pRb phosphorylation and decreased levels of cyclins A and D and cyclin-dependent kinase 2. Calpain 4 knockout fibroblasts also exhibit impaired v-Src-induced morphological transformation and anchorage-independent growth. Thus, modulation of the calpain-calpastatin proteolytic system plays an important role in focal adhesion disassembly, morphological transformation, and cell cycle progression during v-Src-induced cell transformation.

Topics: Actins; Animals; Calcium-Binding Proteins; Calpain; CDC2-CDC28 Kinases; Cell Division; Cell Size; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Cyclin A; Cyclin D; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Cyclins; Cytoskeleton; Feedback, Physiological; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Genes, myc; Genes, ras; Immunohistochemistry; Mice; Mice, Knockout; Models, Biological; Oncogene Protein p65(gag-jun); Oncogene Protein pp60(v-src); Oncogene Proteins v-fos; Protein Isoforms; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Retinoblastoma Protein; Temperature

Precise control of the level of c-Myc protein is important to normal cellular homeostasis, and this is accomplished in part by degradation through the ubiquitin-proteasome pathway. The calpains are a family of calcium-dependent proteases that play important roles in proteolysis of some proteins, and their possible participation in degradation of intracellular c-Myc was therefore investigated. Activation of calpain with the cell-permeable calcium ionophore A23187 in Rat1a-myc or ts85 cells in culture induced rapid cleavage of c-Myc. This degradation was both calpain- and calcium-dependent since it was inhibited by preincubation with either the calpain-inhibitory peptide calpeptin or the calcium-chelating agent EGTA. A23187-induced c-Myc cleavage occurred in a time-dependent manner comparable to that of FAK, a known calpain substrate, and while calpeptin was able to significantly protect c-Myc from degradation, inhibitors of the proteasome or caspase proteases could not. Exposure of Rat1a-myc or ts85 cells in culture to calpeptin, or to the thiol-protease inhibitor E64d, resulted in the accumulation of c-Myc protein without an impact on ubiquitin-protein conjugates. Using an in vitro assay, calpain-mediated degradation occurred rapidly with wild-type c-Myc as the substrate, but was significantly prolonged in some c-Myc mutants with increased transforming activity derived from lymphoma patients. Those mutants with a prolonged half-life in vitro were also more resistant to A23187-induced cleavage in intact cells. These studies support a role for calpain in the control of c-Myc levels in vivo, and suggest that mutations impacting on sensitivity to calpain may contribute to c-Myc-mediated tumorigenesis.

Topics: Animals; Blotting, Western; Calcimycin; Calcium; Calpain; Cell Line; Cell Transformation, Neoplastic; Chelating Agents; Cysteine Endopeptidases; Dipeptides; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Ionophores; Lymphoma; Mammary Neoplasms, Experimental; Mice; Multienzyme Complexes; Mutation; Peptide Hydrolases; Proteasome Endopeptidase Complex; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-myc; Rats

The purpose of this study is to indicate that oxidative stress may contribute to occurrence of meningiomas. Recently, it was reported that aside from the neurofibromatosis type 2 (NF2) gene mutations, the calpain-dependent proteolysis of the NF2 gene product, merlin might be closely related to the development of certain NF2-related tumors. Although meningiomas are well known to occur more frequently in aged persons, it still remains unknown why calpain activation occurs predominantly in them. Because the production of free radicals with aging might be one of the causes of calpain activation especially in leptomeningeal cells being devoid of blood supply, the authors examined the relations between mu-calpain activation and merlin proteolysis induced by the oxidative stress.. The authors examined 12 patient-derived sporadic meningiomas and their primary cultured cells. Malignant glioma cell line (U-251MG), which had no relation to NF2, was used as a control. They were exposed to hydrogen peroxide (H2O2) for 1 hour. After oxidative stress, they were examined by Western blot and immunofluorescence microscopic analyses.. Despite the consistent expressions of activated mu-calpain in 11 of 12 meningioma tissues, this calpain activation completely disappeared after culture; instead the full-length merlin appeared again in 8 of 11 cases. The treatment of cultured cells with hydrogen peroxide induced both mu-calpain-dependent cleavage of merlin and reduction of an intrinsic calpain inhibitor calpastatin. Such proteolysis was significantly blocked by a specific calpain inhibitor, Z-LLal. The full-length merlin was immunocytochemically colocalized with activated mu-calpain at the plasma membrane, and, after mu-calpain activation, the fragment of merlin translocated to the perinuclear cytoplasm or into the nucleus.. These findings suggest that oxidative stress-induced activation of mu-calpain causes proteolysis of merlin conceivably to impair cell adhesion and/or contact inhibition of meningioma cells.

Topics: Brain Neoplasms; Calpain; Cell Adhesion; Cell Transformation, Neoplastic; Female; Humans; Hydrogen Peroxide; Male; Meningioma; Middle Aged; Neurofibromin 2; Oxidants; Oxidative Stress; Tumor Cells, Cultured

We previously have described the use of an antisense RNA strategy termed random homozygous knock-out (RHKO) to identify negative regulators of cell proliferation. Here we report the discovery that RHKO-mediated deficiency of the nCL-4 calpain protease results in cellular transformation of and tumorigenesis by murine NIH3T3 fibroblasts. We isolated cell clones able to form colonies on 0.5% soft agar and found that these cells generated tumors when injected subcutaneously into nude mice. The gene inactivated by RHKO was identified as nCL-4 by genomic library screening, transcript analysis, and DNA sequencing. Anchorage-independent growth, as indicated by colony formation on soft agar, was reversed by reversal of antisense-mediated homozygous inactivation, but continued haplo-insufficiency of nCL-4 resulting from insertional mutagenesis of one nCL-4 allele was associated with persistent tumorigenesis. nCL-4 cDNA expressed in naive 3T3 cells in the antisense, but not sense, direction under control of the cytomegalovirus early promoter reproduced the anchorage-independent growth effects of RHKO. Our results implicate deficiency of the nCL-4 calpain protease in neoplastic transformation.

Topics: 3T3 Cells; Alleles; Animals; Base Sequence; Blotting, Southern; Blotting, Western; Calpain; Cell Division; Cell Transformation, Neoplastic; Cytomegalovirus; DNA, Complementary; Female; Gene Library; Mice; Mice, Knockout; Mice, Nude; Models, Genetic; Molecular Sequence Data; Mutagenesis, Site-Directed; Neoplasm Transplantation; Neoplasms; Peptides; Promoter Regions, Genetic; Recombinant Fusion Proteins; RNA, Antisense; RNA, Messenger; Sequence Analysis, DNA