Proteinase suppression by E-cadherin-mediated cell-cell attachment in premalignant oral keratinocytes

The expression and activity of epithelial proteinases is under stringent control to prevent aberrant hydrolysis of structural proteins and disruption of tissue architecture. E-cadherin-dependent cell-cell adhesion is also important for maintenance of epithelial structural integrity, and loss of E-cadherin expression has been correlated with enhanced invasive potential in multiple tumor models. To address the hypothesis that there is a functional link between E-cadherin and proteinase expression, we have examined the role of E-cadherin in proteinase regulation. By using a calcium switch protocol to manipulate junction assembly, our data demonstrate that initiation of de novo E-cadherin-mediated adhesive contacts suppresses expression of both relative matrix metalloproteinase-9 levels and net urinary-type plasminogen activator activity. E-cadherin-mediated cell-cell adhesion increases both phosphatidylinositol 3'-kinase (PI3-kinase)-dependent AKT phosphorylation and epidermal growth factor receptor-dependent MAPK/ERK activation. Pharmacologic inhibition of the PI3-kinase pathway, but not the epidermal growth factor receptor/MAPK pathway, prevents E-cadherin-mediated suppression of proteinases and delays junction assembly. Moreover, inhibition of junction assembly with a function-blocking anti-E-cadherin antibody stimulates proteinase-dependent Matrigel invasion. As matrix metalloproteinase-9 and urinary-type plasminogen activator potentiate the invasive activity of oral squamous cell carcinoma, these data suggest E-cadherin-mediated signaling through PI3-kinase can regulate the invasive behavior of cells by modulating proteinase secretion.     

Ultraviolet irradiation increases FADD protein in apoptotic human keratinocytes

Ultraviolet irradiation (UV) can induce keratinocyte apoptosis by activating death receptors that recruit the intracellular adaptor molecule FADD/MORT1 (Fas-associating death domain protein/mediator of receptor-induced toxicity). We hypothesized that UV could alter FADD expression levels to augment UV-induced keratinocyte apoptosis. In a dose-dependent manner UV B irradiation increased the expression of FADD protein in a human keratinocyte cell line (CCD-1106) with a corresponding increase in caspase-8 cleavage and cellular apoptosis. FADD overexpression induced cell death in 80% of cells compared with 10% spontaneous cell death in controls. Inhibition of FADD protein by adenoviral expression of anti-sense FADD reduced keratinocyte apoptosis. Regulation of FADD expression by UV may serve to enhance death receptor-mediated keratinocyte death.     

Distribution of actin filaments in human malignant keratinocytes

Distribution of actin filaments in human malignant keratinocytes was examined by immunofluorescence staining. The primary cultures were obtained from a squamous cell carcinoma, a basal cell carcinoma, and Bowen's disease. Rhodamine-phalloidin staining revealed that actin filaments were occasionally organized to form stress fibers, many short bundles with a ripple appearance, and regular arrays of actin patches. Some of these structures appeared in untransformed keratinocytes as a result of a brief exposure to a tumor promotor, TPA. These findings suggest that regulation of actin functions is involved in neoplastic processes from the very early stages and that alteration is persistent in neoplastic cells.     

Differential effects of arsenic on calcium signaling in primary keratinocytes and malignant (HSC-1) cells

Arsenic is highly toxic to living cells, especially skin, and skin cancer is induced by drinking water containing arsenic. The molecular mechanisms of arsenic-induced cancer, however, are not well understood. To examine the initial processes in the development of arsenic-induced cancer, we analyzed calcium signaling at an early stage of arsenic treatment of human primary cells and compared the effects with those observed with arsenic treatment in carcinoma-derived cells. We found that arsenic inhibited inositol trisphosphate receptor (IP3R) function in the endoplasmic reticulum by inducing phosphorylation, which led to decreased intracellular calcium levels. Blockade of IP3R phosphorylation by the serine/threonine protein kinase Akt inhibitor wortmannin rescued calcium signaling. In contrast, arsenic treatment of cells derived from a carcinoma (human squamous carcinoma; HSC-1) for 1h had no obvious effect. Taken together, these results suggest that arsenic-induced reduction in calcium signaling is one of the initial mechanisms underlying the malignant transformation in the development of skin cancer.     

Genetic and environmental factors in cutaneous malignant melanoma

Cutaneous malignant melanoma (CMM) is an interesting example of multifactorial disease, where both genetic and environmental factors are involved and interact. Major risk factors include a personal and familial history of melanoma, cutaneous and pigmentary characteristics, sun exposure and reactions to sun exposure. Phenotypic risk factors are likely to be genetically determined. Two high-risk melanoma susceptibility genes-CDKN2A and CDK4-have been identified to date, with a third gene p14(ARF) also being suspected of playing a role. Other high-risk genes are anticipated by the existence of 9p21-unlinked families. A low-risk melanoma-susceptibility gene-MC1R-has also been identified. Current studies aim to identify other susceptibility genes as well as to determine the respective contributions and interactions of the various genetic and environmental factors of CMM and associated phenotypes.     

Selective cytostatic and cytotoxic effects of avermectins]

A natural avermectin complex, aversectin C, was shown to be capable of exerting selective cytostatic effect. It killed proliferating neuroblastoma B 103 cells but was non-toxic for differentiated cells of this culture. The activity of aversectin C was related neither to activation of the GABA alpha-receptors nor to their blocking and was at a large extent due to the action of avermectin A1, a component of aversectin C.     

A characterization of gamma-glutamyltranspeptidase in normal, perinatal, premalignant and malignant rat liver

gamma-Glutamyltranspeptidase displays the following order of activity in tissues of the Fischer 344 rat: kidney much greater than small intestine much greater than cerebral cortex = testis greater than lung much greater than liver = heart. The activity of the hepatic enzyme in rats is: 4-fold higher in females than males; 4-fold higher in male Wistar, Sprague-Dawley and Zucker rats than male Fischer 344 rats; increased 10-fold in very old vs young male Fischer 344. The hepatic enzyme displays significant species variation: the mouse and rat liver enzymes are similar and low in activity, while duck, dog, pig and beef enzymes are 7, 13, 86 and 92-fold higher, respectively, in activity than the male Fischer rat liver enzyme. A liver plasma membrane isolation procedure has been devised which selects for the sinusoidal face of the liver parenchymal cell as assessed by marker enzyme analysis: for these plasma membranes the purification of gamma-glutamyltranspeptidase is 21.5 and the recovery is 42% indicating that this is the cellular and subcellular locus of the enzyme in rat liver. The characteristics of the liver plasma membrane from female rats are: pH optimum of 8.0; classical Michaelis-Menten kinetics; Km of 1.43 mM and Vmax of 33.3 nmol X mg-1 X min-1. In Fischer 344 rats, gamma-glutamyltranspeptidase activities are elevated over adult levels in perinatal liver: in fetal liver homogenates and plasma membranes the activities are increased 179 and 109-fold, respectively. The activity peaks just after birth and declines rapidly over the first 15 postnatal days. The activity of the liver enzyme in the male Fischer 344 rat exhibits a progressive increase throughout diethylnitrosamine-induced hepatocarcinogenesis: it is increased 7.8-fold in homogenates and 5.4-fold in plasma membranes at the early premalignant stage; 74-fold in homogenates and 31-fold in plasma membranes at the later hyperplastic nodular premalignant stage; and 174-fold in homogenates and 61-fold in plasma membranes at the hepatoma stage. The gradual drop in purification during hepatocarcinogenesis is associated with the appearance of the enzyme in the blood.     

Oxidant-induced vascular endothelial growth factor expression in human keratinocytes and cutaneous wound healing

Neutrophils and macrophages, recruited to the wound site, release reactive oxygen species by respiratory burst. It is commonly understood that oxidants serve mainly to kill bacteria and prevent wound infection. We tested the hypothesis that oxidants generated at the wound site promote dermal wound repair. We observed that H(2)O(2) potently induces vascular endothelial growth factor (VEGF) expression in human keratinocytes. Deletion mutant studies with a VEGF promoter construct revealed that a GC-rich sequence from bp -194 to -50 of the VEGF promoter is responsible for the H(2)O(2) response. It was established that at microm concentrations oxidant induces VEGF expression and that oxidant-induced VEGF expression is independent of hypoxia-inducible factor (HIF)-1 and dependent on Sp1 activation. To test the effect of NADPH oxidase-generated reactive oxygen species on wound healing in vivo, Rac1 gene transfer was performed to dermal excisional wounds left to heal by secondary intention. Rac1 gene transfer accelerated wound contraction and closure. Rac1 overexpression was associated with higher VEGF expression both in vivo as well in human keratinocytes. Interestingly, Rac1 gene therapy was associated with a more well defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. Overall, the histological data indicated that Rac1 might be an important stimulator of various aspects of the repair process, eventually enhancing the wound-healing process as a whole. Taken together, the results of this study indicate that wound healing is subject to redox control.     

Two-mutation model for carcinogenesis: joint analysis of premalignant and malignant lesions.

A model for carcinogenesis that postulates two rate-limiting events for malignant transformation is a generalization of the recessive oncogenesis hypothesis, according to which inactivation of homologous tumor suppressor genes leads to cancer. This model has been shown to be consistent with a large body of epidemiologic and experimental data and has recently been used for the analysis of altered hepatic foci in rodents. These foci are considered to be premalignant lesions. In this paper the necessary mathematics for the joint analysis of premalignant and malignant lesions are developed within the framework of this model.     

Stereotaxic fine needle aspiration cytology of clinically occult malignant and premalignant breast lesions

Stereotaxic fine needle aspiration (FNA) cytology was used to study clinically occult (nonpalpable) breast lesions in 114 consecutive patients with mammographically suspicious findings prior to excisional biopsy. The aspirate contained insufficient material for cytologic evaluation in 15 cases (13.2%), which were histologically diagnosed as benign (7 cases), atypical hyperplasia (7 cases) or carcinoma in situ (1 case). The cytologic findings indicated a benign lesion in 77 cases (67.5%), which were histologically diagnosed as benign (71 cases) or atypical ductal hyperplasia (6 cases). The cytologic sample showed atypia in eight cases (7.0%), which were histologically diagnosed as severe atypical ductal hyperplasia (three cases), carcinoma in situ (one case) or proliferative fibrocystic disease (four cases). In the eight cases (7.0%) cytologically interpreted as probably malignant, histology confirmed six invasive carcinomas, one carcinoma in situ and one fibrocystic disease. Of six cases (4.4%) cytologically reported as malignant, five were histologically diagnosed as invasive carcinoma and one as carcinoma in situ. Overall, stereotaxic FNA cytology reported as malignant or probably malignant 14 of the 15 cases with a histologic confirmation of malignancy, for a sensitivity of 93.3%. Cytology correctly identified 78 of the 83 histologically negative cases, for a specificity of 94.0%. The 16 cases histologically diagnosed as ductal hyperplasia, which carries a high risk for subsequent malignancy, were studied in detail in an effort to define histologic and cytologic criteria for this entity. Using selected histologic criteria, 11 of these cases were graded as showing mild-to-moderate atypical hyperplasia and 5 as showing severe atypical hyperplasia. Three of the latter cases were similarly identified by an analogous cytologic grading; the other two cases had insufficient cytologic samples. The total results in this series of 114 cases support the use of stereotaxic FNA cytology in the diagnosis of these nonpalpable breast lesions, examples of which are illustrated. In particular, it may help to raise the low specificity yielded by mammography alone, which would represent a significant advance for the patient in terms of the accuracy, expediency and reduced cost of diagnosing these lesions.     

Expression of heat shock proteins in premalignant and malignant urothelial lesions of bovine urinary bladder

Abnormal heat shock protein (HSP) levels have been observed in a number of human tumours, where they are involved in all hallmarks of cancer. Since bovine urothelial tumours share striking morphological and biochemical features with their human counterparts, the aim of this study was to evaluate the immunohistochemical levels of Hsp27, Hsp60, Hsp72, Hsp73 and Hsp90 in 28 normal bovine urinary bladders and 30 bovine papillomavirus-positive urothelial tumours (9 in situ carcinomas, 9 low-grade and 12 high-grade carcinomas) and adjacent premalignant lesions obtained from cows suffering from chronic enzootic haematuria, in order to investigate the role of these proteins in the process of urothelial carcinogenesis. A semi-quantitative method was used for the analysis of the results. Western blot analysis was also used to confirm HSP expression in normal controls. All investigated HSPs were expressed in normal bovine urothelium, showing characteristic patterns of immunolabelling throughout urothelial cell layers, which usually appeared to be conserved in urothelial hyperplasia and dysplasia. On the other hand, gradual loss of Hsp27 immunostaining resulted to be significantly associated with increasing histological grade of malignancy (P < 0.01). As well, a significantly reduced immunosignal of Hsp73 and Hsp90 was observed in high-grade and low-/high-grade carcinomas, respectively (P < 0.01). In contrast, Hsp60 (P < 0.01) and Hsp72 (P < 0.05) immunoreactivity appeared to be significantly increased both in premalignant and malignant lesions when compared to that observed in normal urothelium, thus suggesting an early involvement of these proteins in neoplastic transformation of urinary bladder mucosa.     

Different requirements for the cytostatic and apoptotic effects of type I interferons. Induction of apoptosis requires ARF but not p53 in osteosarcoma cell lines

The regulation of cell growth is one of the most important effects of type I interferons (IFNs). This response may involve a cytostatic effect or the induction of apoptosis depending on the cell context. Often the growth-inhibitory response of type I IFNs is studied in tumor cell lines carrying mutations of tumor suppressor genes, and therefore, the growth-inhibitory effect can be influenced by inactivation of these important regulators of cell proliferation. In this report, we explored the role of the ARF-p53 pathway in the growth-inhibitory effect of type I IFNs. We found that p53 is only induced in cells that express p14(ARF) (p19(ARF) in mouse cells). Surprisingly, mouse embryonal fibroblasts that are null for p19(ARF) or P53, even after transformation with oncogenic RAS, respond as well as wild type to the growth-inhibitory effect of type I IFNs. Similarly, human ARF(-/-) U2OS and P53(-/-) SAOS-2 cells show a significant decrease in cell proliferation. However, only SAOS-2 or U2OS reconstituted with inducible p14(ARF) undergo apoptosis in response to IFN beta treatment, and this effect was not inhibited by expression of dominant negative p53. These data suggest that (i) at least in specific cell types, the induction of apoptosis by type I IFNs requires an ARF pathway that is p53-independent and (ii) the cytostatic and pro-apoptotic effects of type I IFNs employ different pathways.     

Expression analysis of PMP22/Gas3 in premalignant and malignant pancreatic lesions.

PMP22 is a structural protein of Schwann cells, but it also influences cell proliferation. In the present study, quantitative RT-PCR (QRT-PCR) and immunohistochemistry were used to determine PMP22 mRNA levels and to localize PMP22 in the normal pancreas (n=20), chronic pancreatitis (CP) (n=22), pancreatic ductal adenocarcinoma (PDAC) (n=31), intraductal papillary mucinous neoplasms (IPMN) (n=9), mucinous cystic tumors (MCN) (n=4), and in a panel of PanIN lesions (n=29). PMP22 mRNA levels were significantly higher in CP (3-fold) and PDAC (2.5-fold), compared to normal pancreatic tissues. PMP22 expression was restricted to nerves in the normal pancreas, while in CP and PDAC PMP22 was also expressed in PanIN lesions and in a small percentage of pancreatic cancer cells. PMP22 was weak to absent in the tumor cells of IPMNs and MCNs. PMP22 mRNA was present at different levels in cultured pancreatic cancer cells and up-regulated by transforming growth factor (TGF)-beta1 in 2 of 8 of these cell lines. In conclusion, PMP22 expression is present in both CP and PDAC tissues. Its expression in PanIN lesions and some pancreatic cancer cells in vitro and in vivo suggests a role of PMP22 in the neoplastic transformation process from the normal pancreas to pre-malignant lesions to pancreatic cancer.     

Retinoids alter the direction of differentiation in primary cultures of cutaneous keratinocytes

The effects of vitamin A on the morphological expression of differentiation were studied in cell cultures of cutaneous keratinocytes from the newborn rat. The cells were first cultivated in a medium containing 0.11 mM calcium until a confluent monolayer had been formed. Stratification and terminal differentiation were then triggered by raising the calcium concentration of the medium to 1.96 mM ('normal' culture). The rise in the concentration of calcium was coupled with the addition of retinol (RL) of retinoic acid (RAC) to the medium to produce an excess of vitamin A (high-retinoid culture). Delipidized serum was used to produce a deficiency of vitamin A (low-retinoid culture). The tissue organization and the ultrastructure of the keratinocytes in the stratified culture were the same as those seen in conventional cultures and skin explants. These stratified cultures expressed the morphological features of the epidermis of intact skin. The addition of RL or RAC to the medium enhanced features characteristic of the secretory epithelium, such as the formation of an extensive endoplasmic reticulum, an enlargement of the Golgi zone, and an increase in the number of vacuoles. At the same time, the addition of retinoids diminished features characteristic of the terminal differentiation of the stratified squamous epithelium, such as stratification and keratinization. Deficiency of vitamin A in the medium resulted in a culture with many differentiated layers. The differentiated cells of the low-retinoid cultures contained densely packed tonofilaments and synthesized products that reacted with the monoclonal antibody AE2 that is specific for keratin peptides which are markers of epidermal differentiation. In the cell culture system that is presented here, an excess of retinoids redirected epithelial differentiation from a stratifying and keratinizing epithelium towards a secretory epithelium. This system is a useful tool for elucidating the mechanisms responsible for the effect of vitamin A on the differentiation of epithelial cells.