Single-gas and mixed-gas permeation of N2/CH4 in thermally-rearranged TR-PBO membranes and their 6FDA-bisAPAF polyimide precursor studied by molecular dynamics simulations.

High-performance polymers with polybenzoxazole (PBO) structures, formed via thermal rearrangement (TR) of aromatic polyimide precursors, have been developed for gas separation applications. The present work compares the transport of N2 and CH4 in a 6FDA-bisAPAF polyimide precursor and in its TR-PBO derivative using molecular dynamics (MD) simulations. The modelling closely mimicked the experimental approach by transforming a 6FDA-bisAPAF atomistic model into its corresponding TR-PBO structure via a specific algorithm. The densities and void spaces of both precursor and TR polymers were found to compare well to experimental data. An iterative technique was used to obtain the single-gas sorption isotherms of N2 and CH4 at 338.5 K in both polymers over a range of feed pressures up to and exceeding 65 bar. CH4 was systematically found to be more soluble than N2. Solubilities in both matrices were quite similar with those in TR-PBO being slightly higher due to its larger fraction of significant volume. Volume dilation analyses confirmed a higher resistance to plasticization for TR-PBO. Extended single-gas N2 and CH4 simulations and 2 : 1 binary CH4/N2 mixed-gas simulations were then conducted in both matrices at 338.5 K and at a pressure of ∼65 bar corresponding to natural gas processing conditions. Mixed-gas sorption was modelled using a modification of the aforementioned iterative method, which fixed the pressure and iterated to convergence the number of molecules of each type of penetrant. The gas diffusion coefficients were estimated using the Trajectory-Extending Kinetic Monte Carlo (TEKMC) procedure. As found experimentally, significantly higher diffusivities and permeabilities were observed in the TR polymer, which led to a slightly lower ideal N2/CH4 permselectivity for TR-PBO (∼2.6) when compared to its 6FDA-bisAPAF precursor (∼3.8). However, both models showed a reduced N2/CH4 separation efficiency under 2 : 1 binary CH4/N2 mixed-gas conditions bordering on the loss of selectivity. For 6FDA-bisAPAF, both permeabilities decreased in the mixed-gas case, but more for N2 than for CH4. For TR-PBO, the permeability of the faster N2 decreased while the permeability of the slower CH4 increased under mixed-gas conditions. This confirms that single-gas simulations are not sufficient for the prediction of the actual mixed-gas permselectivity behaviour in such polymers.

Generation of a tissue-specific transgenic model for K8 phosphomutants: A tool to investigate the role of K8 phosphorylation during skin carcinogenesis in vivo.

Keratin 8/18, the predominant keratin pair of simple epithelia, is known to be aberrantly expressed in several squamous cell carcinomas (SCCs), where its expression is often correlated with increased invasion, neoplastic progression, and poor prognosis. The majority of keratin 8/18 structural and regulatory functions are governed by posttranslational modifications, particularly phosphorylation. Apart from filament reorganization, cellular processes including cell cycle, cell growth, cellular stress, and apoptosis are known to be orchestrated by K8 phosphorylation at specific residues in the head and tail domains. Even though deregulation of K8 phosphorylation at two significant sites (Serine73 /Serine431 ) has been implicated in neoplastic progression of SCCs by various in vitro studies, including ours, it is reported to be highly context-dependent. Therefore, to delineate the precise role of Kereatin 8 phosphorylation in cancer initiation and progression, we have developed the tissue-specific transgenic mouse model expressing Keratin 8 wild type and phosphodead mutants under Keratin 14 promoter. Subjecting these mice to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-mediated skin carcinogenesis revealed that Keratin 8 phosphorylation may lead to an early onset of tumors compared to Keratin 8 wild-type expressing mice. Conclusively, the transgenic mouse model developed in the present study ascertained a positive impact of Keratin 8 phosphorylation on the neoplastic transformation of skin-squamous cells.

Role of Electron Microscopy in Early Detection of Altered Epithelium During Experimental Oral Carcinogenesis.

Early detection of altered epithelium can help in controlling the further progression by timely intervention. Alterations in cellular adhesion are one of the hallmarks of cancer progression, which can be detected at the intracellular level using high-resolution electron microscopy. This study aimed to evaluate the role of electron microscopy in the establishment of ultrastructural markers for early detection of altered epithelium using tissues from 4-Nitroquinoline-1-Oxide (4NQO) induced rat tongue carcinogenesis. Our previous study using light microscopy displayed no histopathological alterations in 4NQO treated tissues until 40 days of treatment, while dysplasia, papilloma and carcinoma were detected at 80/120, 160 and 200 days, respectively. However, electron microscopy detected alterations such as detachment of desmosomes from cell membranes and their clustering in the cytoplasm, increased tonofilaments, keratohyaline granules and thickened corneum in 40 days treated corresponding tissues. These alterations are apparent with hyperkeratosis/hyperplasia but remained undetected using light microscopy. Further, in dysplasia, papilloma and carcinoma, gradual and significant loss of desmosomes, leading to the significant widening of intercellular spaces, was observed using iTEM software. These parameters may serve as indicators for progression of oral cancer. Our results highlight the importance of electron microscopy in the early detection of subcellular changes in the altered epithelium.

Aberrant expression of vimentin predisposes oral premalignant lesion derived cells towards transformation.

OBJECTIVE: We have previously reported the aberrant expression of vimentin in human oral premalignant lesions and a 4-Nitroquinoline 1-oxide (4NQO) model of rat lingual carcinogenesis. Hence, we wanted to understand whether the expression of vimentin in early stage contributes to the process of transformation. STUDY DESIGN: Vimentin was stably expressed in oral premalignant lesion derived cells (vimentin negative) and various transformation related phenotypic assays were performed. Since vimentin alone failed to transform the cells, an additional carcinogenic stimulus benzo[a]pyrene (BP) was used. Concomitantly, immunohistochemistry (IHC) was performed on oral leukoplakia and tumor tissues for studying the expression of vimentin and E-cadherin. RESULTS: Exogenous expression of vimentin led to the appearance of EMT and stemness-related signatures. Further, upon BP treatment, vimentin expressing clones showed an increase in vitro and in vivo transformation efficiency. Importantly, high vimentin-low E-cadherin expression significantly correlated with the grade of dysplasia, as also with the lymph node metastasis in oral tumors. CONCLUSION: Our study suggests that the expression of vimentin in early stages may be beneficial, although not sufficient to achieve transformation. Further, high vimentin-low E-cadherin expression, if validated in more number of early oral lesions, may prove useful in the identification of high risk human premalignant lesions.

Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through ß4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating ß4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells.

Alterations in desmosomal adhesion at protein and ultrastructure levels during the sequential progressive grades of human oral tumorigenesis.

With the aim of developing early diagnostic/prognostic markers for oral cancer, desmosomal adhesion in sequentially progressive grades of tissues from oral normal/disorders (normal, hyperplastic, dysplastic, non-metastatic/metastatic tumours, and metastatic nodes) was investigated at protein and ultrastructural levels using immunohistochemistry and transmission electron microscopy, respectively. The expression of desmosomal proteins was higher in hyperplastic tissues than in normal tissues but was significantly decreased in subsequent progressive stages of the disease. Altered expression of desmosomal proteins was significantly correlated with local recurrence and disease-free survival. Ultrastructural analysis in the corresponding tissues revealed cytoplasmic clustering of desmosomes in hyperplasia; in more advanced disease stages, a significantly lower number of desmosomes and widened intercellular spaces were observed. Altered protein expression resulting in structural changes was confirmed by knocking down desmoplakin expression in non-transformed cells, which failed to form normal desmosome structures and induced a cell-transformation phenotype. Our data suggest that alterations in desmosomal assembly initiate at an early hyperplastic grade and, with more advanced disease stages, the severity of the alterations gradually becomes higher. Alterations in desmosomal adhesion can be useful for early detection of high-risk premalignant lesions, as well as for identification of invasive characteristics of primary non-metastatic tumours. Early detection will help to control further progression of disease by timely intervention.

Quantitative phosphoproteomic analysis reveals system-wide signaling pathways regulated by site-specific phosphorylation of Keratin-8 in skin squamous cell carcinoma derived cell line.

Keratin 8/18, a simple epithelia specific keratin pair, is often aberrantly expressed in squamous cell carcinomas (SCC) where its expression is correlated with increased invasion and poor prognosis. Majority of Keratin 8 (K8) functions are governed by its phosphorylation at Serine73 (head-domain) and Serine431 (tail-domain) residues. Although, deregulation of K8 phosphorylation is associated with progression of different carcinomas, its role in skin-SCC and the underlying mechanism is obscure. In this direction, we performed tandem mass tag-based quantitative phosphoproteomics by expressing K8 wild type, phosphodead, and phosphomimetic mutants in K8-deficient A431 cells. Further analysis of our phosphoproteomics data showed a significant proportion of total phosphoproteome associated with migratory, proliferative, and invasive potential of these cells to be differentially phosphorylated. Differential phosphorylation of CDK1T14,Y15 , EIF4EBP1T46,T50 , EIF4BS422 , AKT1S1T246,S247 , CTTN1T401,S405,Y421 , and CAP1S307/309 in K8-S73A/D mutant and CTTN1T401,S405,Y421 , BUB1BS1043 , and CARHSP1S30,S32 in K8-S431A/D mutants as well as some anonymous phosphosites including MYCS176 , ZYXS344 , and PNNS692 could be potential candidates associated with K8 phosphorylation mediated tumorigenicity. Biochemical validation followed by phenotypic analysis further confirmed our quantitative phosphoproteomics data. In conclusion, our study provides the first global picture of K8 site-specific phosphorylation function in neoplastic progression of A431 cells and suggests various potential starting points for further mechanistic studies.

Functional Implications of O-GlcNAcylation-dependent Phosphorylation at a Proximal Site on Keratin 18.

Keratins 8/18 (K8/18) are phosphoglycoproteins and form the major intermediate filament network of simple epithelia. The three O-GlcNAcylation (Ser(29), Ser(30), and Ser(48)) and two phosphorylation (Ser(33) and Ser(52)) serine sites on K18 are well characterized. Both of these modifications have been reported to increase K18 solubility and regulate its filament organization. In this report, we investigated the site-specific interplay between these two modifications in regulating the functional properties of K18, like solubility, stability, and filament organization. An immortalized hepatocyte cell line (HHL-17) stably expressing site-specific single, double, and triple O-GlcNAc and phosphomutants of K18 were used to identify the site(s) critical for regulating these functions. Keratin 18 mutants where O-GlcNAcylation at Ser(30) was abolished (K18-S30A) exhibited reduced phosphorylation induced solubility, increased stability, defective filament architecture, and slower migration. Interestingly, K18-S30A mutants also showed loss of phosphorylation at Ser(33), a modification known to regulate the solubility of K18. Further to this, the K18 phosphomutant (K18-S33A) mimicked K18-S30A in its stability, filament organization, and cell migration. These results indicate that O-GlcNAcylation at Ser(30) promotes phosphorylation at Ser(33) to regulate the functional properties of K18 and also impact cellular processes like migration. O-GlcNAcylation and phosphorylation on the same or adjacent sites on most proteins antagonize each other in regulating protein functions. Here we report a novel, positive interplay between O-GlcNAcylation and phosphorylation at adjacent sites on K18 to regulate its fundamental properties.

14-3-3γ-Mediated transport of plakoglobin to the cell border is required for the initiation of desmosome assembly in vitro and in vivo.

The regulation of cell-cell adhesion is important for the processes of tissue formation and morphogenesis. Here, we report that loss of 14-3-3γ leads to a decrease in cell-cell adhesion and a defect in the transport of plakoglobin and other desmosomal proteins to the cell border in HCT116 cells and cells of the mouse testis. 14-3-3γ binds to plakoglobin in a PKCµ-dependent fashion, resulting in microtubule-dependent transport of plakoglobin to cell borders. Transport of plakoglobin to the border is dependent on the KIF5B-KLC1 complex. Knockdown of KIF5B in HCT116 cells, or in the mouse testis, results in a phenotype similar to that observed upon 14-3-3γ knockdown. Our results suggest that loss of 14-3-3γ leads to decreased desmosome formation and a decrease in cell-cell adhesion in vitro, and in the mouse testis in vivo, leading to defects in testis organization and spermatogenesis.

Prognostic role of Oct4, CD44 and c-Myc in radio-chemo-resistant oral cancer patients and their tumourigenic potential in immunodeficient mice.

OBJECTIVE: In the present study, we have investigated the prognostic value of known stem cell-associated molecules such as Oct4, CD44 and c-Myc in patients with oral SCC who had received post-surgery radio- and/or chemotherapy. MATERIALS AND METHODS: Immunohistochemistry was performed to analyse the expression of Oct4, CD44 and c-Myc in 87 tumour tissues, and the expression profile obtained was correlated with clinicopathological parameters of the patients with oral cancer. Tumourigenic potential of these molecules was also evaluated by in vivo studies. RESULTS: Our results showed significant correlation of Oct4 (OS, p = 0.003; DFS, p = 0.001) and c-Myc (OS, p = 0.01; DFS, p = 0.03) with overall survival and disease-free survival independently. Furthermore, all the three markers in combinations of two markers each, i.e. Oct4 + CD44 (OS, p = 0.003; DFS, p = 0.001), Oct4 + c-Myc (OS, p = 0.0001; DFS, p = 0.0001), CD44 + c-Myc (OS, p = 0.008; DFS, p = 0.02) and in combinations of three markers each, i.e. Oct4 + CD44 + c-Myc (OS, p = 0.0001; DFS, p = 0.0001) also significantly correlated with overall survival and disease-free survival. Univariate and multivariate analyses further established the independent prognostic value of Oct4. Oct4-, CD44- and c-Myc-enriched populations independently induced sarcomatoid carcinomas whereas primary keratinocytes developed poorly differentiated carcinomas in immunodeficient mice. CONCLUSIONS: Oct4 and c-Myc independently as well as in combination with CD44 might be useful for the prediction of local recurrence and poor survival of patients with oral cancer which is the novel finding of this study. CLINICAL RELEVANCE: Oct4, c-Myc and CD44 can be used to predict local recurrence and the outcome of treatment in oral cancer patients. In addition, these molecules may find use as molecular targets for effective therapy.

Loss of keratins 8 and 18 leads to alterations in α6ß4-integrin-mediated signalling and decreased neoplastic progression in an oral-tumour-derived cell line.

Keratins 8 and 18 (K8 and K18) are predominantly expressed in simple epithelial tissues and perform both mechanical and regulatory functions. Aberrant expression of K8 and K18 is associated with neoplastic progression and invasion in squamous cell carcinomas (SCCs). To understand the molecular basis by which K8 promotes neoplastic progression in oral SCC (OSCC), K8 expression was inhibited in AW13516 cells. The K8-knockdown clones showed a significant reduction in tumorigenic potential, which was accompanied by a reduction in cell motility, cell invasion, decreased fascin levels, alterations in the organization of the actin cytoskeleton and changes in cell shape. Furthermore, K8 knockdown led to a decrease in α6ß4 integrin levels and α6ß4-integrin-dependent signalling events, which have been reported to play an important role in neoplastic progression in epithelial tissues. Therefore, modulation of α6ß4 integrin signalling might be one of the mechanisms by which K8 and K18 promote malignant transformation and/or progression in OSCCs.

A Molecular Dynamics Study of Single-Gas and Mixed-Gas N2 and CH4 Transport in Triptycene-Based Polyimide Membranes.

Fluorinated polyimides incorporated with triptycene units have gained growing attention over the last decade since they present potentially interesting selectivities and a higher free volume with respect to their triptycene-free counterparts. This work examines the transport of single-gas and mixed-gas N2 and CH4 in the triptycene-based 6FDA-BAPT homopolyimide and in a block 15,000 g mol-1/15,000 g mol-1 6FDA-mPDA/BAPT copolyimide by using molecular dynamics (MD) simulations. The void-space analyses reveal that, while the free volume consists of small-to-medium holes in the 6FDA-BAPT homopolyimide, there are more medium-to-large holes in the 6FDA-mPDA/BAPT copolyimide. The single-gas sorption isotherms for N2 and CH4 over the 0-70 bar range at 338.5 K show that both gases are more soluble in the block copolyimide, with a higher affinity for methane. CH4 favours sites with the most favourable energetic interactions, while N2 probes more sites in the matrices. The volume swellings remain limited since neither N2 nor CH4 plasticise penetrants. The transport of a binary-gas 2:1 CH4/N2 mixture is also examined in both polyimides under operating conditions similar to those used in current natural gas processing, i.e., at 65.5 bar and 338.5 K. In the mixed-gas simulations, the solubility selectivities in favour of CH4 are enhanced similarly in both matrices. Although diffusion is higher in 6FDA-BAPT/6FDA-mPDA, the diffusion selectivities are also close. Both triptycene-based polyimides under study favour, to a similar extent, the transport of methane over that of nitrogen under the conditions studied.

Fascin overexpression promotes neoplastic progression in oral squamous cell carcinoma.

BACKGROUND: Fascin is a globular actin cross-linking protein, which plays a major role in forming parallel actin bundles in cell protrusions and is found to be associated with tumor cell invasion and metastasis in various type of cancers including oral squamous cell carcinoma (OSCC). Previously, we have demonstrated that fascin regulates actin polymerization and thereby promotes cell motility in K8-depleted OSCC cells. In the present study we have investigated the role of fascin in tumor progression of OSCC. METHODS: To understand the role of fascin in OSCC development and/or progression, fascin was overexpressed along with vector control in OSCC derived cells AW13516. The phenotype was studied using wound healing, Boyden chamber, cell adhesion, Hanging drop, soft agar and tumorigenicity assays. Further, fascin expression was examined in human OSCC samples (N = 131) using immunohistochemistry and level of its expression was correlated with clinico-pathological parameters of the patients. RESULTS: Fascin overexpression in OSCC derived cells led to significant increase in cell migration, cell invasion and MMP-2 activity. In addition these cells demonstrated increased levels of phosphorylated AKT, ERK1/2 and JNK1/2. Our in vitro results were consistent with correlative studies of fascin expression with the clinico-pathological parameters of the OSCC patients. Fascin expression in OSCC showed statistically significant correlation with increased tumor stage (P = 0.041), increased lymph node metastasis (P = 0.001), less differentiation (P = 0.005), increased recurrence (P = 0.038) and shorter survival (P = 0.004) of the patients. CONCLUSION: In conclusion, our results indicate that fascin promotes tumor progression and activates AKT and MAPK pathways in OSCC-derived cells. Further, our correlative studies of fascin expression in OSCC with clinico-pathological parameters of the patients indicate that fascin may prove to be useful in prognostication and treatment of OSCC.

Alterations in keratins and associated proteins during 4- Nitroquinoline-1-oxide induced rat oral carcinogenesis.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the sixth largest group of malignancies globally and the single largest group of malignancies in the Indian subcontinent. Despite the advances in treatment and therapeutic modalities the five year survival rate of OSCC has not changed in the last few decades, and remains less than 40%. Several studies have focused on defining molecular markers that can either detect cancer at an early stage or can predict patient's outcome. However, such markers are still undefined. Keratins (K) are epithelia predominant intermediate filament proteins which are expressed in a differentiation dependent and site specific manner. Keratins are being used as biomarkers in different epithelial disorders including cancer. They are associated with desmoplakin and α6ß4 integrin which are components of desmosomes and hemidesmosomes respectively. MATERIALS AND METHODS: 4-Nitroquinoline 1-Oxide (4NQO) was used as a carcinogen for the development of various stages of oral carcinogenesis in rat lingual mucosa. Two-Dimentional gel electrophoresis was performed for the separation of Keratins followed by western blotting for their specific identification. Western blotting and RT PCR was carried out for desmoplakin and α6ß4 integrin respectively to understand their levels. Immunohistochemical analysis was carried out to further study the localization of desmoplakin and α6 integrin. RESULTS: In this study we have analysed the alterations in Keratins and associated proteins during sequential stages of 4NQO induced rat oral carcinogenesis. Our results showed that the alterations primarily begin after the dysplastic changes in the lingual epithelium like the elevation of Keratins 5/6a, ectopic expression of Keratin 8, increase in suprabasal expression of α6 integrin and increase in desmoplakin levels. Most of these alterations persisted till the development of SCC except desmoplakin, the levels of which were downregulated in papillomatous lesions and SCC. Many of these alterations have also been documented in human oral carcinogensis. CONCLUSION: Thus, 4NQO model of rat lingual carcinogenesis reproduces majority of the changes that are seen in human oral carcinogenesis and it can be exploited for the development of biomarkers.

Utility of Keratins as Biomarkers for Human Oral Precancer and Cancer.

Human oral cancer is the single largest group of malignancies in the Indian subcontinent and the sixth largest group of malignancies worldwide. Squamous cell carcinomas (SCC) are the most common epithelial malignancy of the oral cavity, constituting over 90% of oral cancers. About 90% of OSCCs arise from pre-existing, potentially malignant lesions. According to WHO, OSCC has a 5-year survival rate of 45-60%. Late diagnosis, recurrence, and regional or lymph nodal metastases could be the main causes of the high mortality rates. Biomarkers may help categorize and predict premalignant lesions as high risk of developing malignancy, local recurrence, and lymph nodal metastasis. However, at present, there is a dearth of such markers, and this is an area of ongoing research. Keratins (K) or cytokeratins are a group of intermediate filament proteins that show paired and differentiation dependent expression. Our laboratory and others have shown consistent alterations in the expression patterns of keratins in both oral precancerous lesions and tumors. The correlation of these changes with clinicopathological parameters has also been demonstrated. Furthermore, the functional significance of aberrant keratins 8/18 expression in the malignant transformation and progression of oral tumors has also been documented. This article reviews the literature that emphasizes the value of keratins as biomarkers for the prognostication of human oral precancers and cancers.

Biomarker Potential of Vimentin in Oral Cancers.

Oral carcinogenesis is a multistep process. As much as 5% to 85% of oral tumors can develop from potentially malignant disorders (PMD). Although the oral cavity is accessible for visual examination, the ability of current clinical or histological methods to predict the lesions that can progress to malignancy is limited. Thus, developing biological markers that will serve as an adjunct to histodiagnosis has become essential. Our previous studies comprehensively demonstrated that aberrant vimentin expression in oral premalignant lesions correlates to the degree of malignancy. Likewise, overwhelming research from various groups show a substantial contribution of vimentin in oral cancer progression. In this review, we have described studies on vimentin in oral cancers, to make a compelling case for vimentin as a prognostic biomarker.

O-GlcNAcylation determines the solubility, filament organization, and stability of keratins 8 and 18.

Keratins 8 and 18 (K8/18) are intermediate filament proteins expressed specifically in simple epithelial tissues. Dynamic equilibrium of these phosphoglycoproteins in the soluble and filament pool is an important determinant of their cellular functions, and it is known to be regulated by site-specific phosphorylation. However, little is known about the role of dynamic O-GlcNAcylation on this keratin pair. Here, by comparing immortalized (Chang) and transformed hepatocyte (HepG2) cell lines, we have demonstrated that O-GlcNAcylation of K8/18 exhibits a positive correlation with their solubility (Nonidet P-40 extractability). Heat stress, which increases K8/18 solubility, resulted in a simultaneous increase in O-GlcNAc on these proteins. Conversely, increasing O-GlcNAc levels were associated with a concurrent increase in their solubility. This was also associated with a notable decrease in total cellular levels of K8/18. Unaltered levels of transcripts and the reduced half-life of K8 and K18 indicated their decreased stability on increasing O-GlcNAcylation. On the contrary, the K18 glycosylation mutant (K18 S29A/S30A/S48A) was notably more stable than the wild type K18 in Chang cells. The K18-O-GlcNAc mutant accumulated as aggregates upon stable expression, which possibly altered endogenous filament architecture. These results strongly indicate the involvement of O-GlcNAc on K8/18 in regulating their solubility and stability, which may have a bearing on the functions of these keratins.

Multifaceted role of keratins in epithelial cell differentiation and transformation.

Keratins, the epithelial-predominant members of the intermediate filament superfamily, are expressed in a pairwise, tissuespecific and differentiation-dependent manner. There are 28 type I and 26 type II keratins, which share a common structure comprising a central coiled coil α-helical rod domain flanked by two nonhelical head and tail domains. These domains harbor sites for major posttranslational modifications like phosphorylation and glycosylation, which govern keratin function and dynamics. Apart from providing structural support, keratins regulate various signaling machinery involved in cell growth, motility, apoptosis etc. However, tissue-specific functions of keratins in relation to cell proliferation and differentiation are still emerging. Altered keratin expression pattern during and after malignant transformation is reported to modulate different signaling pathways involved in tumor progression in a context-dependent fashion. The current review focuses on the literature related to the role of keratins in the regulation of cell proliferation, differentiation and transformation in different types of epithelia.

Cytokeratin fragments in the serum: their utility for the management of oral cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy and is a major cause of cancer morbidity and mortality worldwide. Oral cancer is the most predominant malignancy in the Indian subcontinent due to the widespread habits of chewing tobacco and related products. Patients with oral tumours have a high risk of early locoregional relapse. Early detection of disease progression remains a challenging task mainly due to the lack of adequate early prognostic markers. CEA, SCC Ag, CA-125, serum cytokeratin (CK) fragments, Cyfra 21-1 (CK 19), TPS (CK 18), TPA (CK 8, 18, and 19) etc. are being used as serum markers for the prediction of prognosis of various malignancies. This review presents the available literature on serum CK markers in different malignancies evaluates their utility in the management of oral cancer, and identifies the lacunae which need to be addressed to develop sensitive and specific assays for early detection of recurrence, prognosis, and treatment monitoring.

Keratin 5/14­mediated cell differentiation and transformation are regulated by TAp63 and Notch­1 in oral squamous cell carcinoma­derived cells.

Keratins 5/14 (K5/14) are intermediate filament proteins expressed in the basal layer of stratified epithelial cells and are known targets of p63. Previous research in our laboratory showed that upon K5/14 downregulation in oral squamous cell carcinoma (OSCC)­derived cells, there was an increase in intracellular Notch­1 levels and differentiation markers such as involucrin, keratin 1 and a decrease in tumorigenic potential in vivo. However, the molecules involved in the K14 regulated cell differentiation and transformation are not known to date. In order to understand the possible role of TAp63, we downregulated TAp63 in a K14­knockdown background. We observed that there was a decrease in the expression of Notch­1. Expression levels of differentiation markers such as involucrin, K1, loricrin and filaggrin were also decreased. Furthermore, TAp63 downregulation led to an increase in invasion, migration and in vivo tumorigenic potential of these cells. We observed a decrease in ß­catenin signaling in K14­downregulated cells. Notably, when TAp63 was downregulated in K14­knockdown cells, there was increase in non­phospho ß­catenin levels. Hence, this study indicates that TAp63 plays an important role in K14­downregulated cells possibly by regulating the Notch­1 expression. K14 regulates the expression of TAp63 which in turn regulates expression of Notch­1. The present study is a step forward in our quest to understand the functional significance of molecules that regulate the process of differentiation and tumorigenesis in stratified epithelial cells.