The role of CD44 in the development and prognosis of head and neck squamous cell carcinomas.

CD44, the product of a single gene, exists as several isoforms generated by alternative exon splicing and posttranslational modifications, and is widely distributed in different cells and tissues including those of squamocellular origin. CD44 is a cell surface glycoprotein involved in many cellular processes acting as a receptor for cell to cell or cell to matrix adhesion, as a signal transmitter and as a growth factor-presenting molecule. Numerous studies based on immunohistochemical analyses of paraffin-embedded or frozen tissue sections using different monoclonal antibodies to CD44 isoforms and molecular biological techniques have provided evidence that in many types of tumours there is overexpression of CD44 isoforms and aberrant processing of immature CD44 transcripts relative to non-neoplastic control tissues, suggesting a role of CD44 in tumour development and progression. In contrast to these malignancies, one or more of the CD44 splice-variant isoforms are down-regulated in squamous cell carcinomas of the head and neck. CD44-deficient mice develop normally without giving rise to spontaneous tumours, but CD44-negative cells appear to be more susceptible to oncogenic transformation. Reduction in the expression of CD44 may confer growth advantage and malignant properties to tumour cells. The clinical significance of CD44 in squamous cell carcinomas of the head and neck as a tumour marker for cancer diagnosis and prognosis is discussed.

Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece.

Clusterin/apolipoprotein J is a novel biomarker of cellular senescence that does not affect the proliferative capacity of human diploid fibroblasts.

Normal human fibroblasts have a limited replicative potential in culture and eventually reach a state of irreversible growth arrest, termed senescence. In a previous study aiming to identify genes that are differentially regulated during cellular senescence we have cloned clusterin/apolipoprotein J (Apo J), a 80 kDa secreted glycoprotein. In the current report we pursue our studies and show that senescence of human diploid fibroblasts is accompanied by up-regulation of both Apo J mRNA and protein levels, but with no altered biogenesis, binding partner profile or intracellular distribution of the two Apo J forms detected. To analyze the causal relationship between senescence and Apo J protein accumulation, we stably overexpressed the Apo J gene in primary as well as in SV40 T antigen-immortalized human fibroblasts and we showed no alteration of the proliferative capacity of the transduced cells. Despite previous reports on tumor-derived cell lines, overexpression of Apo J in human fibroblasts did not provide protection against apoptosis or growth arrest induced by hydrogen peroxide. Overall, our results suggest that Apo J overexpression does not induce senescence but it is rather a secondary consequence of the senescence phenotype. To our knowledge this is the first report that provides a functional analysis of human Apo J during replicative senescence.

Chondrocyte phenotype and cell survival are regulated by culture conditions and by specific cytokines through the expression of Sox-9 transcription factor.

OBJECTIVE: To investigate the effects of culture conditions, serum and specific cytokines such as insulin-like growth factor (IGF) 1 and interleukin (IL) 1alpha on phenotype and cell survival in cultures of Syrian hamster embryonic chondrocyte-like cells (DES4(+).2). METHODS: Proteins and RNA extracted from subconfluent and confluent early- and late-passage DES4(+).2 cells cultured in the presence or absence of serum and IL-1alpha or IGF-1 or both cytokines together were analysed for the expression of chondrocyte-specific genes and for the chondrogenic transcription factor Sox-9 by Western and Northern blotting. Apoptosis was assessed by agarose gel electrophoresis of labelled low-molecular weight DNA extracted from DES4(+).2 cells and another Syrian hamster embryonic chondrocyte-like cell line, 10W(+).1, cultured under the different conditions and treatments. RESULTS: Early passage DES4(+).2 cells expressed chondrocyte-specific molecules such as collagen types alpha1(II) and alpha1(IX), aggrecan, biglycan and link protein and collagen types alpha1(I) and alpha1(X) mRNAs, suggesting a prehypertrophic chondrocyte-like phenotype. The expression of all genes investigated was cell density- and serum-dependent and was low to undetectable in cell populations from later passages. Early-passage DES4(+).2 and 10W(+).1 cells survived when cultured at low cell density, but died by apoptosis when cultured at high cell density in the absence of serum or IGF-1. IGF-1 and IL-1alpha had opposite and antagonistic effects on the chondrocyte phenotype and survival. Whereas IL-1alpha acting alone suppressed cartilage-specific gene expression without significantly affecting cell survival, IGF-1 increased the steady-state mRNA levels and relieved the IL-1alpha-induced suppression of all the chondrocyte-specific genes investigated; it also enhanced chondrocyte survival. Suppression of the chondrocyte phenotype by the inflammatory cytokine IL-1alpha correlated with marked down-regulation of the transcription factor Sox-9, which was relieved by IGF-1. The expression of the Sox9 gene was closely correlated with the expression of the chondrocyte-specific genes under all conditions and treatments. CONCLUSIONS: The results suggest that the effects of cartilage anabolic and catabolic cytokines IGF-1 and IL-1alpha on the expression of the chondrocyte phenotype are mediated by Sox-9. As Sox-9 appears to be essential for matrix production, the potent effect of IL-1alpha in suppressing Sox-9 expression may limit the ability of cartilage to repair during inflammatory joint diseases.

Induction of gynogenesis and androgenesis in goldfish Carassius auratus (var. oranda).

Chromosome engineering was used to develop lines that expressed the stable phenotypic characteristics of goldfish, Carassius auratus (var. oranda). To obtain androgenetic individuals, carp eggs were irradiated and fertilized with sperm from goldfish bearing telescopic eyes. The fertilized eggs were divided into four groups and three of them were subjected to heat-shock treatment at 42 degrees C for 2.0 min at 34, 37 and 40 min after fertilization. All groups exhibited low viability of embryos and after hatching the embryos were severely deformed on the head, yolk sac and tail and exhibited 100% mortality, within 2 days after hatching. To obtain gynogenetic individuals, a thin layer of carp milt was subjected to ultraviolet irradiation for 26 min to neutralize its genetic material and used to fertilize a mixture of eggs derived from three different variations of C. auratus individuals with characteristics such as round fat body, triple tail, red and black colour and telescopic eyes. The fertilized eggs were divided into six groups and five of them were subjected to heat-shock treatment at 39.5 degrees C for 2.5 min at 15 (group 2), 20 (group 3), 30 (group 5) and 45 (group 6) min after fertilization. The percentage of fertilized eggs that survived ranged from 20 to 50% and the percentage of larval survival in groups 2-5 was 22-28% with lower levels in groups 1 and 6, ranging from 10-12%. However, 40-60% of the larvae exhibited severe deformities on the head, yolk sac and tail, whereas the rest developed normally. Fish with the typical oranda phenotype were observed in all the groups and about 1% of the fish were characterized by a triple tail but there was no fish with telescopic eyes. The results indicate that gynogenetic individual goldfish can be produced but that the induction of androgenesis requires further improvement of the techniques.

v-FBR-fos oncogene fails to rescue mammalian cells from growth arrest but affects the responses of human fibroblasts to heparin.

The effects of v-fos oncogene on the proliferation of mammalian cells were studied using several approaches. Constitutive overexpression of v-FBR-fos in normal human fibroblasts (MRC-5) and of v-FBR-fos in human chondrocytes (HAC21) failed to immortalise them, extend their in vitro lifespan, increase their growth rates or induce cellular transformation. Further, v-FBR-fos did not render MRC-5 growth factor-independent or alter their responsivenness to serum, but it markedly suppressed their heparin-induced proliferation. A conditionally immortalized, temperature-sensitive rat embryo fibroblast cell line (tsa14) which undergoes growth arrest upon inactivation of a thermolabile SV40 large T antigen by a temperature shift producing a phenotype that mimmicks the senescent phenotype, was also used to study the effects of v-FBR-fos on cell proliferation. Whereas a wild-type SV40 large T antigen rescued tsa14 from a temperature-dependent growth arrest, v-FBR-fos failed to do so. Hence, v-FBR-fos was not sufficient to, at least, complement the tsa14 growth defect. There was no change in the expression of c-jun and junB, members of the AP-1 transcriptional complex in MRC-5v-fos cells. These data show that v-FBR-fos is not sufficient to rescue mammalian cells from senescence but it can affect the responses of human fibroblasts to heparin suggesting a role of fos in cell proliferation.

Prognostic significance of p53 in the cancer of the larynx.

p53 is a nuclear phosphoprotein acting as a transcription factor to regulate cell cycle progression and apoptosis, mediated by a number of target genes. p53 mutant proteins have lost a) the ability to act as sequence-specific transcription factors and b) their tumour suppressive properties. As p53 is the most commonly mutated gene in human cancer, including laryngeal squamous cell carcinoma, an aggressive and most frequent tumour of head and neck, it has attracted a great deal of interest as a prognostic factor, diagnostic tool and therapeutic target. This article reviews the current understanding of the prognostic significance of p53 in laryngeal squamous cell carcinoma. Immunohistochemical staining techniques and molecular genetics demonstrated that p53 activation is an early event in laryngeal squamous cell carcinogenesis but can not be used as a reliable prognostic marker.

Suppression of decorin expression and partial induction of anchorage-independent growth by the v-src oncogene in human fibroblasts.

To determine its role in cell transformations, the v-src oncogene was introduced into the human foetal diploid fibroblasts MRC-5 and into MRC-SV1, a simian virus 40 (SV40)-transformed cell line derived from them. Infected cells were found to contain stably integrated intact proviruses, as determined by Southern blot analysis. Although highly expressed, v-src did not change the morphology or growth patterns of MRC-5 cells and failed to induce foci or alter their saturation densities. However, overexpression of v-src reduced the plating efficiencies of MRC-5 and induced anchorage-independent growth in a low but significant number of cells. Northern blot analysis showed that v-src selectively abolished the expression of decorin, a small dermatan/chondroitin sulphate proteoglycan that interacts with extracellular-matrix components and modulates collagen-fibril formation and the activity of transforming growth factor (TGF) beta1. Addition of herbimycin A, a potent pp60src tyrosine-kinase inhibitor, resulted in the reexpression of decorin in MRC-5 carrying v-src. There were no changes in the expressions of fibronectin, procollagen type I, or tissue plasminogen activator, an activator of extracellular-matrix-degrading enzymes. Moreover, v-src did not alter the expressions of the epidermal-growth-factor receptor or TGFbeta1 or reduce the growth-factor requirements of MRC-5 fibroblasts. MRC-5 and MRC-SV1 expressing v-src remained non-tumourigenic when injected into nude mice. Constitutive expression of v-src did not alter the mRNA levels of c-jun and junB, suggesting that the effects of the oncogene are not mediated by AP-1. Decorin gene expression has been shown previously to be maximal in quiescent cells and virtually absent in transformed ones. Our data indicate that the ability to synthesise decorin can be suppressed in human fibroblasts without their becoming transformed, and that the relations between decorin synthesis and growth controls need further clarification.

Modulation of elongation factor-1 delta (EF-1 delta) expression by oncogenes in human epithelial cells.

A cDNA clone covering part of the C-terminal domain of human EF-1 delta was isolated from mammary cancer cells by subtractive hybridisation. The higher expression of EF-1 delta in the tumours suggested that malignant transformation in vivo requires an increase in translation factor mRNA and protein synthesis for entry into and transition through the cell cycle. To explore the relation between cell division and EF-1 delta expression, MCF-7 cells were treated with dexamethasone, an inducer of differentiation. There was no change in the mRNA levels of EF-1 delta in the dexamethasone-treated cells. To explore the relation between oncogenes and EF-1 delta expression, a variety of oncogenes were introduced into human mammary epithelial cells (MCF-7) and human keratinocytes (HaCaT). Despite high oncogene mRNA expression, there was no significant change in the EF-1 delta mRNA level by v-src, c-erbB (EGF Receptor), c-erbB-2, v-myc and v-fos oncogenes. However, overexpression of v-Ha-ras in HaCaT cells resulted in a three to five-fold decrease in the steady-state mRNA level of EF-1 delta. Taken together, the data provides further support on the interaction of translation factors and oncogenic transformation.

Stable expression of CD1a molecule in human epithelial cell lines shows temperature-dependent expression and affects cell morphology and growth.

The human CD1a molecule is a transmembrane protein which shares structural similarities with HLA class I molecules. It has restricted tissue distribution in normal individuals, and is a useful diagnostic marker for certain disease states such as Langerhans cell histiocytosis. In order to investigate the function of this molecule, a cDNA fragment encoding the CD1a molecule was cloned into several EUKARYOTIC expression vectors which were then used to establish human epithelial cell lines stably expressing the membrane-bound CD1a molecule. Human keratinocytes (HaCaT) and epithelial cells (HeLa) stably expressing CD1a were established by retroviral-mediated gene transfer and DNA transfection, respectively. Expression and localization of the CD1A molecule were then confirmed by Northern blot analysis and immunofluorescence methods. CD1a expression appears to have profound effects on cellular growth and morphology. Both stably CD1a-expressing HeLa and HaCaT cells showed increased doubling times, and up to 20% of CD1a-expressing cells showed altered cell morphology. Clonogenicity experiments demonstrated a reduction in colony size and plating efficiency was augmented in CD1a-positive cells when compared with vector-transfected/infected controls. Our findings suggest that CD1A expression may act as a negative growth regulator in these cells in vitro. Furthermore, lower temperatures greatly enhanced the expression of CD1a at both the protein and mRNA levels in a time-dependent fashion. Since the physiological skin temperatures lie well below the core temperature, this observation may have important implications in the study of Langerhans cells in vitro.

SV40-transformation of embryonic human diploid fibroblasts results in multiple molecular changes.

Attempts were made to identify the cellular and molecular changes associated with SV40-transformation of human fibroblasts, MRC-5. SV40-transformed human fibroblasts, MRC-5V1, displayed a polygonal to round morphology, grew slowly, had reduced plating efficiencies but high saturation densities. However, they could be propagated in low serum-containing medium and grew very efficiently in soft agar. These altered growth properties of MRC-5V1 suggested that SV40 induced changes in cell adhesion and growth factor requirements. Indeed, MRC-5V1 expressed markedly reduced levels of cellular fibronectin, high levels of tPA and the expression of procollagen alpha 2(I) and decorin were absent. Moreover, MRC-5V1 did not express HGF/SF, a paracrine effector of epithelial cells and expressed very low levels of EGF receptor. However, SV40 induced the expression of TGF alpha, one of the ligands of the EGF receptor, TGF beta 1 and TGF beta 2, all of which are associated with cellular transformation. Given the establishment of autocrine loops in MRC-5V1 and the fact that decorin interacts with fibronectin and collagens and negatively regulates the activity of TGF beta s, these changes could account for the altered growth and transformation properties of MRC-5V1. Several studies have provided a link between oncogenic transformation, transcriptional and translational control. SV40 markedly reduced the expression of junB but not c-jun in MRC-5V1 and the expression of EIF-4E and EF1 delta were not significantly affected. The data shows that SV40-transformation of human fibroblasts is associated with multiple genetic changes affecting the expression of genes involved in cell adhesion, signal transduction and transcription, hence suggesting the breakdown of several cellular control mechanisms.

Overexpression of the human c-erbB (EGF receptor) protooncogene fails to alter the lifespan or promote tumourigenic growth of normal and SV40-transformed human fibroblasts.

c-erbB was introduced into normal human fibroblasts, MRC-5, which expressed normal levels of EGF receptor and in a SV40-transformed cell line, MRC-5V1, derived from them, which expressed markedly reduced levels of EGF receptor mRNA. MRC-5 overexpressing c-erbB, responded mitogenically to EGF. However, addition of high EGF concentrations markedly reduced DNA synthesis and resulted in the inhibition of cellular growth. In contrast, MRC-5V1 exhibited an increase in DNA synthesis in an EGF-dependent manner which was enhanced by overexpression of c-erbB. These cells, unlike MRC-5, also produced TGF alpha, an EGF receptor ligand which is often associated with cellular transformation. Ligand-activation of EGF receptor did not alter the lifespan, induce focus formation or anchorage-independence of MRC-5 and all the cell types remained non-tumourigenic in nude mice. However, c-erbB induced the expression of tPA, c-jun and junB in both MRC-5 and MRC-5V1. The data suggest that overexpression and activation of c-erbB is unlikely to play a role in immortalisation of human diploid fibroblasts but it may contribute to cellular transformation.

Expression of cartilage-specific molecules is retained on long-term culture of human articular chondrocytes.

Normal human adult articular chondrocytes were used to determine how the chondrocyte phenotype is modulated by culture conditions following long-term culture. We report here for the first time that human articular chondrocytes have a lifespan in the range of 34-37 population doublings. While chondrocytes cultured as monolayers displayed a fibroblastoid morphology and grew faster, those cultured as suspensions over agarose adopted a round morphology and formed clusters of cells reminiscent of chondrocyte differentiation in intact cartilage, with little or no DNA synthesis. These morphologies were independent of the age of the culture. Despite, these morphological differences, however, chondrocytes expressed markers at mRNA and protein levels characteristic of cartilage: namely, types II and IX collagens and the large aggregating proteoglycans, aggrecan, versican and link protein, but not syndecan, under both culture conditions. However, they also expressed type I collagen alpha 1(I) and alpha 2(I) chains. It has been suggested that expression of collagen alpha 1(I) by chondrocytes cultured as monolayers is a marker of the loss of the chondrocyte phenotype. However, we show here, using reverse transcriptase/polymerase chain reaction, that normal fresh intact human articular cartilage expresses collagen alpha 1(I). The data show that following long-term culture human articular chondrocytes retain their differentiated characteristics and that cell shape does not correlate with the expression of the chondrocyte phenotype. It is proposed that loss of the chondrocyte phenotype is marked by the loss of one or more cartilage-specific molecules rather than by the appearance of non-cartilage-specific molecules.

Overexpression of ha-ras oncogene transforms rodent fibroblasts with low-frequency but not human-diploid fibroblasts.

Activated Ha-ras oncogenes were introduced into early passage normal human adult dermal fibroblasts, 149BR and established mouse Swiss 3T3 fibroblasts by retroviral-mediated gene transfer or by DNA transfection, respectively. The presence and expression of Ha-ras oncogenes was followed by Southern and Northern blotting and immunoprecipitation. Overexpression of the human mutant c-Ha-ras1 T24 oncogene in mouse cells induced morphological transformation, focus formation and growth in soft agar with low frequency. Tumourigenicity studies showed that the malignant transformation of these cells by p21Ha-ras T24 oncoprotein was concentration-dependent and it required additional events suggesting that in vitro establishment is not a sufficient prerequisite for tumourigenic conversion by activated Ha-ras oncogenes. In contrast, constitutive expression of the viral Ha-ras oncogene in human diploid fibroblasts failed to immortalise or transform them. All ras-expressing human cells remained flat, anchorage-dependent and non-tumourigenic suggesting that these cells are resistant to transformation by activated oncogenes. The role of Ha-ras oncogenes in the transformation of mammalian cells in discussed.

Mechanisms responsible for the limited lifespan and immortal phenotypes in cultured mammalian cells.

Normal mammalian cells have a limited lifespan in culture and hypotheses explaining cellular senescence usually fall into one of two categories. One of these postulates that random errors or damage accumulate in essential macromolecules and eventually outstrip the cell's capacity for resynthesis and repair. The second considers the changes when immortal clones are produced from normal cells and in particular the lifespans of hybrids when cells of differing growth potentials are fused. These data can be explained by postulating that the mortal phenotype is dominant and that trans-acting growth inhibitors are involved in limiting lifespan. But the results do not indicate if the inhibitors are the primary cause of senescence or a secondary effect induced by quite different initial events. We suggest that normal cells possess proof-reading mechanisms which monitor the accuracy of chromosome segregation and replication and which can induce the synthesis of growth inhibitors when they detect major errors in chromosome metabolism. It is further postulated that random damage accumulates during the growth of normal cells and eventually leads to detectable chromosome changes and the synthesis of inhibitors. Our hypothesis predicts that the emergence of immortal clones will be linked to the absence of active inhibitors and therefore to a loss in the fidelity of chromosome metabolism. Data are quoted which show that in contrast to normal cells, immortal clones have highly irregular karyotypes, amplify segments of their chromosomes, integrate exogenous DNA efficiently, maintain a constant level of 5-methylcytosine residues and have high frequencies of chromosomal aberrations. The mechanism of the proof-reading is unknown, but it may monitor changes in the patterns by which chromosome domains are attached to the nuclear matrix.