Genomic landscape and immune-related gene expression profiling of epithelial ovarian cancer after neoadjuvant chemotherapy.

Platinum-based neoadjuvant chemotherapy followed by interval debulking surgery is an accepted treatment for patients with stage III or IV epithelial ovarian cancer who are not suitable for primary debulking surgery. The identification of suitable adjuvant treatments in these patients is an unmet need. Here, we explore potential genomic characteristics (mutational and immune-associated expression profiles) in a series of patients undergoing neoadjuvant chemotherapy. Tumor samples from biopsy and interval debulking surgery were analyzed for mutational landscape and immune profiling, together with detailed immunohistochemistry using different immune cell markers, and correlated with clinicopathological characteristics and potential response to neoadjuvant chemotherapy. No major differences in the mutational landscape were observed in paired biopsy and surgery samples. Genomic loss of heterozygosity was found to be higher in patients with total/near-total tumor response. The immune gene expression profile after neoadjuvant chemotherapy revealed activation of several immune regulation-related pathways in patients with no/minimal or partial response. In parallel, neoadjuvant therapy caused a significant increase of tumor-infiltrating lymphocyte population abundance, primarily due to an augmentation of the CD8+ T cell population. Remarkably, these changes occurred irrespective of potential homologous recombination defects, such as those associated with BRCA1/2 mutations. Our study strengthens the use of loss of heterozygosity as a biomarker of homologous repair deficiency. The changes of immune states during neoadjuvant chemotherapy reveal the dynamic nature of tumor-host immune interactions and suggest the potential use of immune checkpoint inhibitors or their combination with poly-ADP polymerase inhibitors in high stage and grade epithelial ovarian cancer patients undergoing neoadjuvant therapy.

Macrophage polarization as a novel weapon in conditioning tumor microenvironment for bladder cancer: can we turn demons into gods?

Macrophages are major components of the immune infiltration in cancer where they can affect tumor behavior. In the bladder, they play important roles during the resolution of infectious processes and they have been associated with a worse clinical prognosis in bladder cancer. The present review focused on the characteristics of these important immune cells, not only eliciting an innate immune surveillance, but also on their importance during the cancer immunoediting process. We further discuss the potential of targeting macrophages for anticancer therapy, the current strategies and the state of the art as well as the foreseen role on combined therapies on the near future. This review shows how a comprehensive understanding of macrophages within the tumor should translate to better clinical outcome and new therapeutic strategies focusing especially on bladder cancer.

Progeny of Lgr5-expressing hair follicle stem cell contributes to papillomavirus-induced tumor development in epidermis.

Epidermal keratinocytes and hair follicle (HF) stem cells (SCs) expressing oncogenes are competent at developing squamous cell carcinomas (SCCs) in epidermis and HFs, respectively. To determine whether bulge and hair germ (HG) SCs from HF contribute to SCC generation at distant epidermis, the most frequent epidermal region where these lesions arise in human skin, we used a skin cancer mouse model expressing E6 and E7 oncoproteins from Human papillomavirus (HPV) 16 in SCs and basal keratinocytes. This previously described mouse model recapitulates the human skin papillomavirus-induced SCC pathology. We show that E6 and E7 expression promote the expansion of keratin 15 (K15)-expressing cells. These K15(+) aberrant cells exhibit some HGSC markers and diminished expression of Tcf3 and Sox9 hair SC specification genes, which are accumulated in HFs and mislocalized to interfollicular epidermis. Leucine-rich G-protein-coupled receptor 5 (Lgr5)-expressing SCs, localized in the bulge and HG, are the origin of the expanded K15(+) cell population. A large subset of the Lgr5(+) SC progeny, expressing K15 and P-cadherin, is aberrantly mobilized to the upper region of HFs and the epidermis, and accumulates at E6/E7-induced pre-neoplastic lesions and epidermal tumors. These findings indicate that aberrant accumulation of altered SCs in HFs and their subsequent migration to the epidermis contribute to HPV-induced tumor development.

E2F1 loss induces spontaneous tumour development in Rb-deficient epidermis.

The specific ablation of Rb1 gene in epidermis (Rb(F/F);K14cre) promotes proliferation and altered differentiation but does not produce spontaneous tumour development. These phenotypic changes are associated with increased expression of E2F members and E2F-dependent transcriptional activity. Here, we have focused on the possible dependence on E2F1 gene function. We have generated mice that lack Rb1 in epidermis in an inducible manner (Rb(F/F);K14creER(TM)). These mice are indistinguishable from those lacking pRb in this tissue in a constitutive manner (Rb(F/F);K14cre). In an E2F1-null background (Rb(F/F);K14creER(TM); and E2F1(-/-) mice), the phenotype due to acute Rb1 loss is not ameliorated by E2F1 loss, but rather exacerbated, indicating that pRb functions in epidermis do not rely solely on E2F1. On the other hand, Rb(F/F);K14creER(TM);E2F1(-/-) mice develop spontaneous epidermal tumours of hair follicle origin with high incidence. These tumours, which retain a functional p19(arf)/p53 axis, also show aberrant activation of ß-catenin/Wnt pathway. Gene expression studies revealed that these tumours display relevant similarities with specific human tumours. These data demonstrate that the Rb/E2F1 axis exerts essential functions not only in maintaining epidermal homoeostasis, but also in suppressing tumour development in epidermis, and that the disruption of this pathway may induce tumour progression through specific alteration of developmental programs.

The orphan receptor GPR55 drives skin carcinogenesis and is upregulated in human squamous cell carcinomas.

G protein-coupled receptors (GPCRs) control crucial physiological processes and their dysfunction contributes to various human diseases, including cancer. The orphan GPCR GPR55 was identified and cloned more than a decade ago, but very little is known about its physio-pathological relevance. It has been recently shown that GPR55 controls the behavior of human cancer cell lines in culture and xenografts. However, the assessment of the actual role of this receptor in malignant transformation in vivo is hampered by the lack of studies on its functional impact in clinically-relevant models of cancer. Here we demonstrate that GPR55 drives mouse skin tumor development. Thus, GPR55-deficient mice were more resistant to DMBA/TPA-induced papilloma and carcinoma formation than their wild-type littermates. GPR55 exerted this pro-tumor effect primarily by conferring a proliferative advantage on cancer cells. In addition, GPR55 enhanced skin cancer cell anchorage-independent growth, invasiveness and tumorigenicity in vivo, suggesting that it promotes not only tumor development but also tumor aggressiveness. Finally, we observed that GPR55 is upregulated in human skin tumors and other human squamous cell carcinomas compared with the corresponding healthy tissues. Altogether, these findings reveal the pivotal importance of GPR55 in skin tumor development, and suggest that this receptor may be used as a new biomarker and therapeutic target in squamous cell carcinomas.

p27Kip1 represses transcription by direct interaction with p130/E2F4 at the promoters of target genes.

The cyclin-cdk (cyclin-dependent kinase) inhibitor p27Kip1 (p27) has a crucial negative role on cell cycle progression. In addition to its classical role as a cyclin-cdk inhibitor, it also performs cyclin-cdk-independent functions as the regulation of cytoskeleton rearrangements and cell motility. p27 deficiency has been associated with tumor aggressiveness and poor clinical outcome, although the mechanisms underlying this participation still remain elusive. We report here a new cellular function of p27 as a transcriptional regulator in association with p130/E2F4 complexes that could be relevant for tumorigenesis. We observed that p27 associates with specific promoters of genes involved in important cellular functions as processing and splicing of RNA, mitochondrial organization and respiration, translation and cell cycle. On these promoters p27 co-localizes with p130, E2F4 and co-repressors as histone deacetylases (HDACs) and mSIN3A. p27 co-immunoprecipitates with these proteins and by affinity chromatography, we demonstrated a direct interaction of p27 with p130 and E2F4 through its carboxyl-half. We have also shown that p130 recruits p27 on the promoters, and there p27 is needed for the subsequent recruitment of HDACs and mSIN3A. Expression microarrays and luciferase assays revealed that p27 behaves as transcriptional repressor of these p27-target genes (p27-TGs). Finally, in human tumors, we established a correlation with overexpression of p27-TGs and poor survival. Thus, this new function of p27 as a transcriptional repressor could have a role in the major aggressiveness of tumors with low levels of p27.

Akt pathway as a target for therapeutic intervention in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common form of cancer worldwide. One frequent alteration found in this type of cancer is overactivation of the PI3K/PTEN/mTOR pathway, of which protein kinase B (PKB)/Akt is a central key element, controlling important cellular processes such as metabolism, cell size, proliferation and apoptosis, ultimately regulating cell growth and survival. Thus, drugs that target Akt directly or elements of the pathway are plausible candidates for cancer treatment. Accordingly, numerous clinical trials in various phases are being performed for these drugs. In this review, we discuss the tumorigenic capacity of Akt and focus on its role in HNSCC, paying special attention to the current efforts in treating this cancer in a more specific, Akt-targeted way, based on its primordial role in this type of cancer.

Molecular determinants of Akt-induced keratinocyte transformation.

The PI3K/PTEN/Akt signaling pathway has emerged in recent years as a main player in human cancers, increasing proliferation and decreasing apoptosis of transformed cells, and thus becoming a potential target for therapeutic intervention. Our previous data have demonstrated that Akt-mediated signaling is of a key relevance in the mouse skin carcinogenesis system, one of the best-known models of experimental carcinogenesis. Here, we investigated the involvement of several pathways as mediators of Akt-induced increased proliferation and tumorigenesis in keratinocytes. Tumors produced by subcutaneous injection of Akt-transformed keratinocytes showed increased Foxo3a phosphorylation, but no major alterations in p21(Cip1/WAF1), p27(Kip1) or mdm2 expression and/or localization. In contrast, we found increased expression and nuclear localization of DeltaNp63, beta-catenin and Lef1. Concomitantly, we also found increased expression of c-myc and CycD1, targets of the beta-catenin/Tcf pathway. Such increase is associated with increased phosphorylation and stabilization of c-myc protein as well as increased translation of c-myc and CycD1 due to mTOR activation. Using immunohistochemistry approaches in samples of oral dysplasias and human head and neck squamous cell carcinomas, we confirmed that increased Akt activation significantly correlates with increased DeltaNp63 and CycD expression, c-myc phosphorylation and nuclear accumulation of beta-catenin. Collectively, these results demonstrate that Akt is able to transform keratinocytes by specific mechanisms involving transcriptional and post-transcriptional processes.

Regulation of the differentiation-related gene Drg-1 during mouse skin carcinogenesis.

Differentiation-related gene-1 (Drg-1) has been identified as a gene whose expression is increased in several processes related to differentiation, but its function is currently unknown. In this report, we show that Drg-1 was expressed in keratinocytes, this expression being rapidly increased as a result of induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) or the presence of an activating form of Ha-ras. Induction by TPA occurred both in cultured cell lines and primary keratinocytes as well as in mouse skin after a single TPA application. Overexpression of Drg-1 was also observed in TPA-induced hyperplastic skin. In agreement, mouse skin papillomas and carcinomas also overexpressed Drg-1. In addition, Drg-1 was induced when keratinocytes were forced to differentiate by calcium switch or serum starvation. Analysis of the expression of Drg-1 during the keratinocyte cell cycle demonstrated relatively high levels of Drg-1 mRNA in G(0), which increased in early G(1) and decreased afterwards in late G(1)/S. In situ analysis showed an accumulation of Drg-1 in the suprabasal layers of the skin, as well as in the more differentiated areas of mouse skin papillomas. These results suggest that, in addition to being upregulated during keratinocyte differentiation, the Drg-1 gene might have a complex function in skin tumorigenesis.

Inhibition of protein kinase B (PKB) and PKCzeta mediates keratin K10-induced cell cycle arrest.

The intermediate filament cytoskeleton is composed of keratins in all epithelial cells and imparts mechanical integrity to these cells. However, beyond this shared function, the functional significance of the carefully regulated tissue- and differentiation-specific expression of the large keratin family of cytoskeletal proteins remains unclear. We recently demonstrated that expression of keratin K10 or K16 may regulate the phosphorylation of the retinoblastoma protein (pRb), inhibiting (K10) or stimulating (K16) cell proliferation (J. M. Paramio, M. L. Casanova, C. Segrelles, S. Mittnacht, E. B. Lane, and J. L. Jorcano, Mol. Cell. Biol. 19:3086-3094, 1999). Here we show that keratin K10 function as a negative modulator of cell cycle progression involves changes in the phosphoinositide 3-kinase (PI-3K) signal transduction pathway. Physical interaction of K10 with Akt (protein kinase B [PKB]) and atypical PKCzeta causes sequestration of these kinases within the cytoskeleton and inhibits their intracellular translocation. As a consequence, the expression of K10 impairs the activation of PKB and PKCzeta. We also demonstrate that this inhibition impedes pRb phosphorylation and reduces the expression of cyclins D1 and E. Functional and biochemical data also demonstrate that the interaction between K10 and these kinases involves the non-alpha-helical amino domain of K10 (NTerm). Together, these results suggest new and essential roles for the keratins as modulators of specific signal transduction pathways.

The ink4a/arf tumor suppressors cooperate with p21cip1/waf in the processes of mouse epidermal differentiation, senescence, and carcinogenesis.

In mammalian cells, cell cycle withdrawal is a prerequisite for terminal differentiation. Accordingly, in most tissues, including epidermis, the expression of the cyclin-dependent kinase inhibitors increases during differentiation. However, the actual role of cyclin-dependent kinase inhibitors is unclear. Different aspects of epidermal growth and differentiation in ink4a(Delta2,3)-null, p21-null, and ink4a(Delta2,3)/p21-doubly deficient mice were studied. Altered differentiation and decreased age-related senescence were found in the epidermis of ink4a(Delta2,3)/p21-null mice and, to a lesser extent, in ink4a(Delta2,3)- and p21-null mice. ink4a(Delta2,3)/p21-null primary keratinocytes underwent cell cycle arrest upon calcium or transforming growth factor-beta treatment, but failed to differentiate. This differentiation deficiency was not observed in p21- or ink4a(Delta2,3)-deficient keratinocytes. Upon infection with a v-Ha-ras-coding retrovirus, wild-type keratinocytes displayed features indicative of premature cell senescence. In p21- or ink4a(Delta2,3)-deficient keratinocytes, only a partial response was observed. ink4a(Delta2,3)/p21-deficient keratinocytes did not display senescent features, but showed increased tumorigenic potential upon injection into nude mice. These results indicate that ink4a/arf and cip1/waf genes cooperate to allow normal keratinocyte differentiation and that the absence of both favors malignant transformation.

Opposite functions for E2F1 and E2F4 in human epidermal keratinocyte differentiation.

Proteins of the retinoblastoma family (pRb, p107, and p130) modulate cell proliferation, a function related to their capacity to control the activity of the E2F transcription factor family. The Rb proteins also control cell differentiation in different tissues. We have recently described their involvement in human epidermal keratinocyte differentiation (Paramio, J. M., Lain, S., Segrelles, C., Lane, E. B. , and Jorcano, J. L. (1998) Oncogene 17, 949-957). Here we show that E2F proteins are also involved in this process. We found that E2F1 and E2F4 are expressed differentially during the in vitro differentiation of human epidermal keratinocytes, with the former uniformly present throughout the process, whereas the second is predominantly expressed at the onset of differentiation. This pattern is also observed in human skin by confocal microscopy. Electrophoretic mobility shift assays and immunoprecipitation experiments demonstrated that the complexes formed by E2F1 and E2F4 and Rb family proteins vary throughout in vitro keratinocyte differentiation. In agreement with this observation, several E2F-responsive genes are differentially regulated during this process. To test the functional implications of these observations, we transfected HaCaT keratinocytes with plasmids coding for E2F1 and E2F4. Transfected cells display opposite in vitro differentiation properties. Although E2F1-transfected cells are unable to differentiate, E2F4-transfected cells show an increased differentiation rate compared with Neo-transfected control cells. Our data demonstrate that the differential and coordinated expression and interaction of E2F and Rb proteins modulate the process of epidermal differentiation and provide clear evidence that members of the E2F family of transcription factors play specific and opposite roles during cell differentiation.

p53 is phosphorylated at the carboxyl terminus and promotes the differentiation of human HaCaT keratinocytes.

The p53 phosphoprotein acts as a tumor-suppressor gene product through the inhibition of cell growth and induction of apoptosis in a transcription-dependent manner. These functions require p53 activation through different biochemical postranslational modifications. Given the relevance of this protein in ultraviolet light-induced carcinogenesis, whose targets are primarily skin keratinocytes, we studied the functions of p53 in epidermal cell differentiation. We selected HaCaT cells, a human keratinocyte cell line bearing point-mutated, transcriptionally inactive, but highly stable p53, which facilitates immunochemical and biochemical analysis. In addition, a reliable in vitro differentiation system has been developed with these cells (Paramio et al. Oncogene 17:949, 1998). We report that during HaCaT differentiation there is a loss of immunoreactivity of p53 against antibodies that specifically recognize epitopes located at the carboxyl terminus of the protein. Because treatment with phosphatase restores this immunoreactivity, we conclude that p53 is phosphorylated at the carboxyl terminus during keratinocyte differentiation. This biochemical modification has been associated with the transcriptional activation of the molecule, and because p53 is involved in differentiation processes in other cell types, we investigated the potential functions of p53 during epidermal differentiation. To this end, we generated HaCaT clones expressing a murine temperature-sensitive p53 (Mp53ts) by transfection because the endogenous p53 is not functional even with phosphorylation. We characterized the expression and effects of the transfected protein in different selected clones. The ultraviolet-light response of these clones was restored, demonstrating the functionality of Mp53ts in these cells. We also observed that, with induction of differentiation, Mp53ts transfected cells differentiate faster than the parental or vector-transfected control cells, demonstrating that p53 promotes epidermal differentiation. The sustained expression of p53 in differentiating cells leads to massive cell death and detachment, a phenomenon that may be similar to epidermal terminal differentiation. In addition, we observed that the expression of p53-dependent genes such as p21waf/cip1 and mdm2 (which are known to participate in epidermal differentiation) increases during HaCaT differentiation, i.e., in a p53-independent manner.

PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein.

The tumour suppressor PTEN, also named MMAC1 or TEP1, is associated with a number of malignancies in human populations. This protein has a dual protein phosphatase activity, being also capable to dephosphorylate phosphatidylinositol 3,4,5 triphosphate. We have studied the mechanism of growth suppression attributable to PTEN. We observed that PTEN overexpression inhibits cell growth in a variety of normal and transformed, human and murine cells. Bromodeoxyuridine (BrdU) incorporation and TUNEL labelling experiments in transiently transfected cells demonstrate that this inhibition is due to a cell cycle arrest rather than induction of apoptosis. Given that PTEN is unable to cause cell growth arrest in retinoblastoma (Rb)-deficient cell lines, we have explored the possible requirement for pRb in the PTEN-induced inhibition of cell proliferation. We found that the co-expression of SV40 antigen, but not a mutant form (which binds exclusively to p53), and cyclin D1/cdk4 are able to overcome the PTEN-mediated growth suppression. In addition, the reintroduction of a functional pRb, but not its relatives p107 or p130, in Rb-deficient cells restores the sensitivity to PTEN-induced arrest. Finally, the hyperphosphorylation of transfected pRb is inhibited by PTEN co-expression and restored by PI-3K co-expression. Accordingly, PTEN gene is mostly expressed, in parallel to Akt, in mid-late G1 phase during cell cycle progression prior to pRb hyperphosphorylation. Finally, we have studied the signal transduction pathways modulated by PTEN expression. We found that PTEN-induced growth arrest can be rescued by the co-expression of active PI-3K and downstream effectors such as Akt or PDK1, and also certain small GTPases such as Rac1 and Cdc42, but not by active Ha-ras, raf or RhoA. Collectively, our data link the tumour suppressor activities of PTEN to the machinery controlling cell cycle through the modulation of signalling molecules whose final target is the functional inactivation of the retinoblastoma gene product.

Modulation of cell proliferation by cytokeratins K10 and K16.

The members of the large keratin family of cytoskeletal proteins are expressed in a carefully regulated tissue- and differentiation-specific manner. Although these proteins are thought to be involved in imparting mechanical integrity to epithelial cells, the functional significance of their complex differential expression is still unclear. Here we provide new data suggesting that the expression of particular keratins may influence cell proliferation. Specifically, we demonstrate that the ectopic expression of K10 inhibits the proliferation of human keratinocytes in culture, while K16 expression appears to promote the proliferation of these cells. Other keratins, such as K13 or K14, do not significantly alter this parameter. K10-induced inhibition is reversed by the coexpression of K16 but not that of K14. These results are coherent with the observed expression pattern of these proteins in the epidermis: basal, proliferative keratinocytes express K14; when they terminally differentiate, keratinocytes switch off K14 and start K10 expression, whereas in response to hyperproliferative stimuli, K16 replaces K10. The characteristics of this process indicate that K10 and K16 act on the retinoblastoma (Rb) pathway, as (i) K10-induced inhibition is hampered by cotransfection with viral oncoproteins which interfere with pRb but not with p53; (ii) K10-mediated cell growth arrest is rescued by the coexpression of specific cyclins, cyclin-dependent kinases (CDKs), or cyclin-CDK complexes; (iii) K10-induced inhibition does not take place in Rb-deficient cells but is restored in these cells by cotransfection with pRb or p107 but not p130; (iv) K16 efficiently rescues the cell growth arrest induced by pRb in HaCaT cells but not that induced by p107 or p130; and (v) pRb phosphorylation and cyclin D1 expression are reduced in K10-transfected cells and increased in K16-transfected cells. Finally, using K10 deletion mutants, we map this inhibitory function to the nonhelical terminal domains of K10, hypervariable regions in which keratin-specific functions are thought to reside, and demonstrate that the presence of one of these domains is sufficient to promote cell growth arrest.

A role for phosphorylation in the dynamics of keratin intermediate filaments.

Keratins undergo highly dynamic events in the epithelial cells that express them. These dynamic changes have been associated with important cell processes. We have studied the possible role of keratin phosphorylation-dephosphorylation processes in the control of these dynamic events. Drugs that affect the protein phosphorylation metabolism (activators or inhibitors of protein kinases or protein phosphatases) have been used in two different dynamic experimental systems. First, the behaviour of keratins after the formation of cell heterokaryons, and second, the assembly of a newly synthesised keratin after transfection into the pre-existing keratin cytoskeleton. The main difference between these two systems stems on the alteration of the amount of keratin polypeptides present in the cells, since in heterokaryons this amount was unaltered whilst in transfection experiments there is an increase due to the presence of the transfected protein. We observed in both systems that the inhibition of protein kinases led to a delayed dynamic behaviour of the keratin polypeptides. On the contrary, the inhibition of protein phosphatases by okadaic acid or the activation of protein kinases by phorbol esters promoted a substantial increase in the kinetics of these processes. Biochemical studies demonstrate that this behavioural changes can be correlated with changes in the phosphorylation state of the keratin polypeptides. As a whole, present results indicate that the highly dynamic properties of the keratin polypeptides can be modulated by phosphorylation.

Differential expression and functionally co-operative roles for the retinoblastoma family of proteins in epidermal differentiation.

Terminal differentiation requires cell cycle withdrawal, suggesting the involvement of negative cell cycle controllers in the process. We have analysed the involvement of the retinoblastoma family of proteins (pRb, p107 and p130) in epidermal proliferation and differentiation. These proteins play key roles as inhibitors of cell cycle progression and are involved in muscle and neuron differentiation. We found that during in vitro differentiation of human HaCaT keratinocytes, pRb, p107 and p130 are sequentially expressed, in contrast to the co-expression observed during cell cycle progression in the same cells. Immunofluorescence studies on skin sections revealed the presence of pRb and p107 in basal and suprabasal cell layers, whilst p130 is restricted to cells already committed to differentiation in the suprabasal compartments. To explore the functional significance of the differential expression of these proteins, transfection experiments were performed in HaCaT keratinocytes. We observed that the forced over-expression of pRb, p107 or p130 individually did not induce differentiation of the transfected cells. However, the co-transfection of pRb and p107 induced the expression of early differentiation markers (keratin k10) and triple transfectants pRb+p107+p130 expressed markers representative of later stages of epidermal differentiation (involucrin). Finally, we observed that these three proteins repress keratinocyte proliferation, although to a different extent (p107>pRb> or =p130). These results indicate that the members of the pRb family play specific, yet coordinated roles during epidermal differentiation, and that the ordered progression along the different stages of this process results from the effects of different combinations of these proteins.

Role of protein kinases in the in vitro differentiation of human epidermal HaCaT cells.

Different chemicals that specifically and selectively inhibit or activate protein kinases have been used to define the possible roles of these enzymes in the different steps of epidermal differentiation. Using HaCaT keratinocytes as a model, and under conditions in which cell proliferation is minimally affected, we found that tyrosine kinase inhibition leads to an inhibition of early (spinous; keratin k10 expression) and late (granulosum; involucrin expression) differentiation processes. cGMP- and cAMP-dependent protein kinases appear to modulate the transition from spinous to granular differentiation, a process which seems to be negatively controlled by protein phosphatases. Finally, enzymes belonging to the protein kinase C family appear to facilitate the transition from spinous to granular differentiation programmes while inhibiting the early steps of epidermal differentiation.

Keratin intermediate filament dynamics in cell heterokaryons reveals diverse behaviour of different keratins.

To study the dynamics of keratin intermediate filaments, we fused two different types of epithelial cells (PtK2 and BMGE+H) and studied how the keratins from the parental cells recombine and copolymerize to form the heterokaryon cytoskeleton. The behaviour of the keratins during this process was followed by immunofluorescence using specific antibodies. After fusion, the parental cytoskeletons undergo a depolymerization process most apparent in the region adjacent to the fusion area. The depolymerized subunits spread throughout the heterokaryon and copolymerize into a new hybrid cytoskeleton. The complete process is very rapid, occurring in 3-4 hours, thus demonstrating the highly dynamic nature of the keratin cytoskeleton. Although newly synthesised subunits contribute to the formation of the hybrid cytoskeleton, the process takes place with similar kinetics in the absence of protein synthesis, showing the dynamic nature of the keratins from pre-existing cytoskeletons. During this process, specific keratins behave differently. Keratins K8, K18, K5 and K10 are mobilised from the parental cytoskeletons and reassemble rapidly into the hybrid cytoskeleton (3-6 hours), whereas K14 requires a substantially longer period (9-24 hours). Thus, different keratins, even when they form part of the same heterodimeric/tetrameric complexes, as is the case for K5 and K14, exhibit different dynamics. This suggests that individual polypeptides or homopolymeric complexes rather than exclusively heterodimeric/ tetrameric subunits, as is currently thought, can also take part in keratin intermediate filament assembly and dynamics. Biochemical analysis performed in the absence of protein synthesis revealed greater amounts of K5 than of K14 in the soluble pool of BMGE+H cells. Crosslinking and immunoprecipitation experiments indicated an excess of monomeric K5, as well as of K5/K14 heterodimers and K5 homodimers in the soluble pool. These results are in agreement with the different dynamic behaviour of these keratins observed in immunofluorescence. On the contrary, the phosphorylation levels of K5 and K14 are similar in both the soluble pool and the polymerized fraction, suggesting that phosphorylation does not play an important role in the different dynamics displayed by these two proteins. In summary, our results demonstrate that, following fusion, the keratin intermediate filament network reshapes rather rapidly and that keratins are highly dynamic proteins, although this mobility depends on each particular polypeptide.