Monique Cadrin

Keratin 8 and 18 loss in epithelial cancer cells increases collective cell migration and cisplatin sensitivity through claudin1 up-regulation.

Background: Loss of keratins 8 and 18 (K8/18) is a hallmark of epithelial-mesenchymal transition (EMT), but its role in tumor progression is unclear. Results: Epithelial cancer cells depleted in K8/18 are more invasive and sensitive to cisplatin through the up-regulation of claudin1. Conclusion: K8/18 loss promotes collective cancer cell migration without inducing EMT. Significance: Cancer cell invasion can arise from K8/18 loss independently of EMT. Keratins 8 and 18 (K8/18) are simple epithelial cell-specific intermediate filament proteins. Keratins are essential for tissue integrity and are involved in intracellular signaling pathways that regulate cell response to injuries, cell growth, and death. K8/18 expression is maintained during tumorigenesis; hence, they are used as a diagnostic marker in tumor pathology. In recent years, studies have provided evidence that keratins should be considered not only as markers but also as regulators of cancer cell signaling. The loss of K8/18 expression during epithelial-mesenchymal transition (EMT) is associated with metastasis and chemoresistance. In the present study, we investigated whether K8/18 expression plays an active role in EMT. We show that K8/18 stable knockdown using shRNA increased collective migration and invasiveness of epithelial cancer cells without modulating EMT markers. K8/18-depleted cells showed PI3K/Akt/NF-κB hyperactivation and increased MMP2 and MMP9 expression. K8/18 deletion also increased cisplatin-induced apoptosis. Increased Fas receptor membrane targeting suggests that apoptosis is enhanced via the extrinsic pathway. Interestingly, we identified the tight junction protein claudin1 as a regulator of these processes. This is the first indication that modulation of K8/18 expression can influence the phenotype of epithelial cancer cells at a transcriptional level and supports the hypothesis that keratins play an active role in cancer progression.

Early perturbations in keratin and actin gene expression and fibrillar organisation in griseofulvin-fed mouse liver

BACKGROUND/AIMS Long-term feeding of mice with a diet containing griseofulvin results in the formation of Mallory bodies, keratin K8 and K18 containing aggregates in hepatocytes. These bodies are biochemically and morphologically identical to the Mallory bodies that emerge in several human liver disorders. The aim of this study was to examine the contribution of K8 and K18 and actin to Mallory body formation. METHODS Mice were fed griseofulvin over a period ranging from 1 day to 20 months. Hepatocyte morphology was monitored by immunocytochemistry, gene expression by Northern and run-off transcription assays, and protein level by Western blotting. RESULTS Griseofulvin feeding induced a series of morphological alterations in hepatocytes that could be grouped into 3 phases: appearance of cholestasis during the first week (phase I), partial hepatocyte recovery at 3 months (phase II), and development of typical Mallory bodies after 3 to 5 months (phase III). All these cellular alterations were associated with perturbations in keratin and actin fibrillar status, coupled with increases in K8, K18 and actin mRNA steady-state level and, in K8 and K18 protein content. The transcriptional activity of the genes was not affected. CONCLUSIONS Perturbations in keratin and actin gene expression and fibrillar organisation constitute early events in the griseofulvin-induced pathological process that in the long-term leads to Mallory body formation. The higher keratin and actin mRNA levels reflect significant increases in mRNA stability taking place at the early phase of griseofulvin intoxication in hepatocytes.

Alteration in molecular structure of cytoskeleton proteins in griseofulvin-treated mouse liver: a pressure tuning infrared spectroscopy study.

Different liver diseases are associated with modifications in hepatocyte cytoskeletal organization and formation of Mallory bodies (MBs). Since the structure of a protein is critical for its function, we studied the changes in the molecular structure of the cytoskeletal protein in the liver from mice fed griseofulvin (GF), which is a good animal model for studying liver disease. Using pressure-tuning infrared spectroscopy we compared the infrared spectra of the cytoskeletal proteins from control liver and griseofulvin treated liver. The results show that the overall structure of the cytoskeletal protein was modified by the griseofulvin treatment. A relative increase in the amount of alpha-helices to beta-sheets was observed in the liver cytoskeleton from the GF-treated mice. Moreover, the random coil and the turn segments were dramatically decreased compared to controls. Pressure-induced modifications including denaturation were irreversible in the control samples whereas they were reversible in the griseofulvin-treated samples. These changes reflect important fundamental modifications in the molecular structure of the cytoskeletal proteins in the griseofulvin-treated hepatocytes. We suggest that these changes are related to the modification of the organization of intermediate filaments and the formation of MBs that occur in the GF-treated liver.

The effects of methylmercury on the cytoskeleton of murine embryonal carcinoma cells

Immunofuorescence staining with antibodies to tubulin and vimentin and staining with phalloidin have been used to examine the effects of methylmercury on the cytoskeleton of embryonal carcinoma cells in culture. Exposure of embryonal carcinoma cells to methylmercury (0.01 to 10 μm) resulted in concentration- and time-dependent disassembly of microtubules in interphase and mitotic cells. These effects were reversible when cultures were washed free of methylmercury. Spindle microtubules were more sensitive than those of interphase cells. Spindle damage resulted in an accumulation of cells in prometaphase/metaphase, which; correlated with a temporary delay in the resumption of normal proliferation rate upon removal of methylmercury. Of the interphase cytoskeletal components, microtubules were the first affected by methylmercury. Vimentin intermediate filaments appeared relatively insensitive to methylmercury, but showed a reorganization secondary to the microtubule disassembly. Actin microfilaments appeared unchanged in cells showing complete absence of microtubules. Our results 1) support previous reports suggesting that microtubules are a primary target of methylmercury, 2) document a differential sensitivity of mitotic and interphase microtubule systems and 3) demonstrate the relative insensitivities of other cytoskeletal components.

Distinctive infrared spectral features in liver tumor tissue of mice: Evidence of structural modifications at the molecular level

Mice were treated with griseofulvin (GF) containing diet or control diet for 12 months. The livers from mice fed griseofulvin showed large tumors that were excised and used for analysis. The infrared spectra from control liver tissue and tumor tissue from GF livers were measured and compared as a function of pressure up to 27 kbar. Many changes in the infrared spectral features of the tumor tissue were observed. Results showed that neoplasm formation involved structural modifications of nucleic acids, lipids, carbohydrates, and proteins in the liver cells, which were detected from the abnormal vibrations of the functional groups in these biomolecules. The amount of glycogen was dramatically decreased in the tumor tissue compared to the control tissue. Important changes in the strength of hydrogen-bondings in the phosphodiester backbone of the nucleic acids and in the C-O groups of tissue proteins and carbohydrates were observed. Stronger interchain interactions and thus close interchain packing among the lipids in the GF liver were evident. These results showed very close similarities with those obtained with other types of tumors such as human colon cancer, suggesting that a common pattern of molecular changes has been identified in neoplastic transformation.

Keratin 20 Serine 13 Phosphorylation Is a Stress and Intestinal Goblet Cell Marker

Keratin polypeptide 20 (K20) is an intermediate filament protein with preferential expression in epithelia of the stomach, intestine, uterus, and bladder and in Merkel cells of the skin. K20 expression is used as a marker to distinguish metastatic tumor origin, but nothing is known regarding its regulation and function. We studied K20 phosphorylation as a first step toward understanding its physiologic role. K20 phosphorylation occurs preferentially on serine, with a high stoichiometry as compared with keratin polypeptides 18 and 19. Mass spectrometry analysis predicted that either K20 Ser13 or Ser14 was a likely phosphorylation site, and Ser13 was confirmed as the phospho-moiety using mutation and transfection analysis and generation of an anti-K20-phospho-Ser13 antibody. K20 Ser13 phosphorylation increases after protein kinase C activation, and Ser13-to-Ala mutation interferes with keratin filament reorganization in transfected cells. In physiological contexts, K20 degradation and associated Ser13 hyperphosphorylation occur during apoptosis, and chemically induced mouse colitis also promotes Ser13 phosphorylation. Among mouse small intestinal enterocytes, K20 Ser13 is pref erentially phosphorylated in goblet cells and undergoes dramatic hyperphosphorylation after starvation and mucin secretion. Therefore, K20 Ser13 is a highly dynamic protein kinase C-related phosphorylation site that is induced during apoptosis and tissue injury. K20 Ser13 phosphorylation also serves as a unique marker of small intestinal goblet cells.

Ethanol-induced phosphorylation of cytokeratin in cultured hepatocytes

We studied the effect of ethanol on the phosphorylation of cytokeratins (CKs) in cultured hepatocytes since CK filaments are regulated by phosphorylation and they are abnormal in alcoholic liver disease. Hepatocytes were obtained from 14-day-old rats and cultured for 48 hrs. The hepatocytes were exposed to ethanol (300 mM) for 30 min. The cells were extracted with the buffer containing Triton X-100. The residual insoluble cytoskeletons were analyzed by two dimensional (2D) gel electrophoresis and autoradiography. 2D gel electrophoresis showed CK 55 and CK 49 or 8 and 18 and actin. The CKs had several isoelectric variants. The most basic spot was the dominant protein which was not phosphorylated. The more acidic spots were phosphorylated. After ethanol treatment, the phosphorylation of CK 55 and CK 49 were markedly increased over controls. We compared these results, with the effect of vasopressin (10 nM), TPA (150 nM) and db-cAMP (1 mM) on the phosphorylation of CKs. Vasopressin and TPA caused the phosphorylation of CK 55 and 49 but db-cAMP did not. The results suggest that CKs are phosphorylated by protein kinase C through the phosphoinositide-linked transduction system activated by ethanol.

Akt isoforms regulate intermediate filament protein levels in epithelial carcinoma cells.

Keratin 8 and 18 are simple epithelial intermediate filament (IF) proteins, whose expression is differentiation‐ and tissue‐specific, and is maintained during tumorigenesis. Vimentin IF is often co‐expressed with keratins in cancer cells. Recently, IF have been proposed to be involved in signaling pathways regulating cell growth, death and motility. The PI3K/Akt pathway plays a pivotal role in these processes. Thus, we investigated the role of Akt (1 and 2) in regulating IF expression in different epithelial cancer cell lines. Over‐expression of Akt1 increases K8/18 proteins. Akt2 up‐regulates K18 and vimentin expression by an increased mRNA stability. To our knowledge, these results represent the first indication that Akt isoforms regulate IF expression and support the hypothesis that IFs are involved in PI3K/Akt pathway.

Effects of methylmercury on retinoic acid-induced neuroectodermal derivatives of embryonal carcinoma cells

Immunofluorescence staining with antibodies to tubulin, neurofilaments and glial filaments was used to study the effects of methylmercury on the differentiation of retinoic acid-induced embryonal carcinoma cells into neurons and astroglia and on the cytoskeleton of these neuroectodermal derivatives. Methylmercury did not prevent undifferentiated embryonal carcinoma cells from developing into neurons and glia. Treatment of committed embryonal carcinoma cells with methylmercury doses exceeding 1 μM resulted in the formation of neurons with abnormal morphologies. In differentiated cultures, microtubules were the first cytoskeletal element to be affected. Their disassembly was time- and concentration-dependent. Microtubules in glial cells and in neuronal perikarya were more sensitive than those in neuronal processes. Neurofilaments and glial filaments appeared relatively insensitive to methylmercury treatment but showed reorganization after complete disassembly of the microtubules. The data demonstrate 1) the sensitivity of microtubules of both neurons and glia to methylmercury-induced depolymerization, and 2) the heterogeneous response of neuronal

Differential phosphorylation of CK8 and CK18 by 12-O-tetradecanoyl-phorbol-13-acetate in primary cultures of mouse hepatocytes

The phosphorylation of cytokeratin was investigated in primary cultures of hepatocytes. The two hepatocyte cytokeratins CK8 and CK18 (55,000 and 49,000 M(r) respectively) were phosphorylated, CK8 being more phosphorylated than CK18. Treatment of the hepatocytes with 150 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA) an activator of protein kinase C induced a transient increase in the level of phosphorylation of CK8 but not CK18. This effect was maximal after 15 min of TPA treatment and was maintained for up to 3 h. After 22 h of treatment with TPA, which down-regulates protein kinase C, CK8 phosphorylation was returned to the basal level. Further addition of TPA to the 22-h treated cells did not cause an increase in CK8 phosphorylation. Indirect immunofluorescence microscopy with a monoclonal antibody to CK8 indicated that while the addition of TPA induced the formation of granular cytokeratin aggregates in some hepatocytes, in most hepatocytes no major changes in the intermediate filament network were observed. Staining for actin showed that actin microfilaments were rapidly reorganized after the treatment and a loss of stress fibres were observed. We propose that CK8 is an in vivo substrate for protein kinase C and that the specific phosphorylation of CK8 plays a role in protein kinase C signal transduction.