TGF-{alpha} and EGF-receptor mRNAs in human oral cancer

Transforming growth factor alpha (TGF-) and epidermal growthfactor receptor (EGFR) have been shown to be present in mostsquamous cell carcinomas. Using the Syrian hamster oral cancermodel, we have recently demonstrated the consistent presenceof TGF- and EGFR mRNAs in chemically transformed hamster oralkeratmocytes. We now present evidence that in human oral cancer(in vivo and in vitro), TGF- and EGFR mRNAs can also be consistentlydetected. No TGF- mRNA can be detected in normal human oralepithelium by Northern blot analysis. These findings reinforcethe use of the hamster cheek pouch as an experimental modelfor the study of oral cancer development, at least in referenceto the possible participation of TGF- in the malignant transformationprocess.     

Differentiation of malignant oral rat keratinocytes reflects changes in EGF and TGF-{beta} receptor expression but not growth factor dependence

This study examined the characteristics of keratinocytes from4-nitroquinoline N-oxide-treated rat oral epithelium that formedwell differentiated carcinomas or spindle cell tumours whentransplanted s.c. to athymic mice. Small polygonal-type cellsin early culture passage gave rise to xenografts that were welldifferentiated carcinomas with keratin positivity and a differentiatedreactivity with two anti-vimentin antibodies (positive Vim Dako,negative Vim 3B4). Progressive culture of the polygonal cellsresulted in cells of a more stellate-like morphology which,when transplanted, formed spindle cell tumours that were keratinnegative and vimentin positive (Vim Dako and Vim 3B4). The stainingpattern of the xenografts was similar to that of the culturedcell derivatives. By contrast to normal oral keratinocytes whichwerestimulated by epidermal growth factor (EGF), the parentaland xenograft-derived cells were markedly less responsive andat times refractory to EGF. Low affinity EGF receptors characterizednormal keratinocytes whilst parental and xenograft-derived cellsfrom well differentiated carcinomass and spindle cell tumoursexpressed diminished numbers of low and high affinity and highaffinity and high affinity EGF receptors respectively. Normalkeratinocytes and parental tumour cells were inhibited by transforminggrowth factor (TGF-ß) whereas xenograft-derived cellLines from both well differentiated carcinomas and spindle celltumours were refractory to TGF-ß. TGF-ßreceptors were predominantly of high affinity although xenograft-derivedcells, particularly from spindle cell tumours, expressed increasednumbers of receptors which were of lower affinity. The resultsindicate that spindle cell tumours are a variant of well differentiatedcarcinomas and express an aberrant pattern of differentiation.Resistance to EGF-induced stimulation and TGF-ß-inducedinhibition appears not to be an integral part of the tumourphenotype but the pattern of EGF and TGF-ß receptorexpression closely follows the degree of tumour differentiation.     

In situ analysis of transforming growth factor-{beta}s (TGF-{beta}1, TGF-{beta}2, TGF-{beta}3and TGF-3 type II receptor expression in malignant melanoma

We have analysed, by in situ hybridization, mRNA expressionof TGF-ß1, TGF-ß2, TGF-ß3, andof TGF-ß type II receptor in benign melanocytic naevi,primary melanomas, and in skin metastases of malignant melanomas.Our results show that melanoma progression correlates with overexpressionof TGF-ß. All skin metastases and most primary melanomasinvasive to Clark's level IV-V revealed specific TGF-ß2mRNA and protein expression. However, expression of this cytokinewas not observed in benign melanocytic lesions and was detectedonly in one of five early primary melanomas investigated. Someprimary melanomas and skin metastases also revealed specificTGF-ß1 mRNA signals although expression of this isoformwas not found in benign naevi. TGF-ß3 expression,which was only barely detectable in benign melanocytic lesions,was enhanced in some skin metastases. Interestingly, the epidermisoverlaying melanomas revealed lower levels of TGF-ß3mRNA expression than epidermis of healthy skin or epidermisadjacent to benign naevi, thereby suggesting that paracrinemechanisms between tumour cells and keratinocytes may influencemelanoma development. In primary melanomas TGF-ß typeII receptor mRNA signals were much more heterogeneously distributedwhen compared to benign melanocytic naevi, suggesting variabledegrees of TGF-ß resistance among melanoma cells withinindividual lesions. However, melanoma progression appeared notto be correlated with a complete loss of TGF-ß typeII receptor gene expression, since all skin metastases revealedclearly detectable although heterogeneous levels of TGF-ßtype II receptor mRNA expression.     

2{alpha} 3{alpha}-Epithio-5{alpha}-androstan-17{beta}-ol in Treatment of Gynecomastia

1. 1. The clinical effect of epitiostanol, a new anti-estrogenagent (2,3-epithio-5a-androstan-17ß-ol) against gynecomastiawas studied in comparison with dromostanolone propionate infifty-four patients ranging from twenty to fifty years in agewithout previous history of hormone therapy and with normalliver function. The experiment was performed for eight weeksby double blind methods in three dosage groups, epithiostanol10 mg, and 20 mg and dromostanolone propionate 50 mg.
2. 2. Epithiostanol20 mg was most effective with regards to effecton mass sizeand tenderness, (effective in 96%, 20/21), followedby 10 mgepitiostanol (effective in 89%, 16/18) and dromostanolonepropionate50 mg (effective in 89%, 16/18) in descending order.No sideeffects were observed in any of the three groups.
3. 3. Basedon the results of the present study, epitiostanol isconcludedto be at least as effective as dromostanolone propionateagainstgynecomastia and to be safe from the viewpoint of sideeffects.A satisfactory therapeutical effect on gynecomastiacan be expectedwith a weekly dosage of 20 mg of epitiostanolfor an administrationperiod of between five to eight weeks.

Present Address: Department of Surgery, Keio University Hospital,Shinanomachi, Shin-juku-ku, Tokyo, Japan.     

Engagement of I-branching {beta}-1, 6-N-acetylglucosaminyltransferase 2 in breast cancer metastasis and TGF-{beta} signaling

In this study, we have showed that GCNT2, a gene-encoding glucosaminyl (N-acetyl) transferase 2, I-branching enzyme, is overexpressed in highly metastatic breast cancer cell lines of human and mouse origin and basal-like breast tumor samples. GCNT2 expression is also significantly correlated to the metastatic phenotype in breast tumor samples. Functional studies showed that ectopic expression of GCNT2 enhances cell detachment, adhesion to endothelial cells, cell migration and invasion in vitro, and lung metastasis of breast cancer cells in vivo. Knockdown of GCNT2 expression decreases cell migration and invasion in vitro and lung metastasis in vivo. We have further shown the involvement of GCNT2 in the epithelial-to-mesenchymal transition (EMT). Specifically, the expression of E-cadherin is significantly changed upon GCNT2 expression at the protein level but not at the RNA level. Moreover, we have shown that GCNT2 is a direct target of the TGF-β-smad pathway and that change in GCNT2 expression modulates EMT induced by TGF-β1 treatment. Finally, we have shown that diminution of the glycosyltransferase activity of I-branching β-1, 6-N-acetylglucosaminyl transferase 2 (GCNT2) abrogates its cell migration and invasion-promoting function and synergistic effect with TGF-β to induce EMT. Our study for the first time showed that GCNT2 is a novel gene contributing to breast cancer metastasis with preferential expression in basal-like breast cancer. Moreover, we discovered that involvement of GCNT2 in EMT and TGF-β signaling, and further glycosylation modification of E-cadherin by GCNT2, are the underlying integrative mechanisms for breast cancer metastasis, implying that blocking TGF-β/GCNT2 signaling is a promising approach for targeting metastatic breast cancer.     

2{alpha},3{alpha}-Epithio-5{alpha}-androstan-17{beta}-yl 1-Methoxycyclopentyl Ether in the Treatment of Advanced Breast Cancer --A Preliminary Clinical Trial--

A new orally active anti-estrogenic steroid, 2,3-epithio-5-androstan-17ß-yl1-methoxycyclopentyl ether (10364-S) was given to 41 advancedbreast cancer patients. Most patients were given a daily doseof 20 mg. The study was preliminary and not a controlled trialusing an already proven androgenic steroid. The remission rateon giving this compound to advanced breast cancer was 11/41or 26.8% and the average duration of the remission was 10.5months. Hoarseness (8/41, 19.5%) and hirsutism (5/41, 12.2%) were relativelyoften seen as virilizing side effects. No unfavorable effectson the hematopoietic organs, the liver or on calcium metabolismwere recognized in the study.     

Effect of {beta}-carotene on cell cycle progression of human fibroblasts

The uptake of ß-carotene (BC) and its effect on thecell cycle progression of normal human fibroblasts in primaryculture were investigated by using two different delivery methods:exposure to BC solubilized in the organic solvent tetrahydrofuran(THF) or to BC incorporated into dipalmitoylphosphatidylcholine(DPPC) liposomes. Cell cycle progression was evaluated by immunofluorescencedetection and flow cytometric analysis of the proliferatingcell nuclear antigen (PCNA). In contrast to THF, which induceda marked reduction in the number of cells in S phase and inthe extent of PCNA immunolabeling, DPPC liposomes proved tobe an effective delivery system that does not interfere withcell proliferation. Cellular uptake of 0.23 nmol/10⁶ cells wasfound after 24 h incubation in BC-containing DPPC liposomes.This value increased to 1.2 nmol/10⁶ cells after 72 h. Afterthe first day of incubation, the number of cells in S phasewas reduced by 50%, with a consequent accumulation of cellsin G₁ phase. This effect was maintained up to 3 days incubation,with no detectable effects on cell viability. This cell cycledelay was found to be reversible, returning the percentage ofcells in S phase to the control value 24 h after removal ofBC from the medium. In order to determine whether the activityof BC could be attributed to the molecule itself or to its conversioninto retinoids, the production of BC metabolites was assessed.Analysis of cellular levels of retinoids failed to demonstratethe presence of retinal, retinol, retinoic acid or retinyl estersduring an incubation period of 6 days. These results suggestthat in normal human fibroblasts, BC induces a cell cycle delayin the G₁ phase and that this effect is independent of conversionto known retinoids.     

Expression of {beta}-catenin, MUC1 and c-met in diffuse-type gastric carcinomas: correlations with tumour progression and prognosis

Previous studies on the immunoreactivity of β-catenin, MUC1 and c-met in gastric carcinomas regarding survival and clinico-pathological features led to contradictory results. Therefore, a series of 94 diffuse-type and mixed-type subcardial gastric carcinomas according to the Laurén classification were investigated to elucidate possible correlations with clinico-pathological and prognostic data. An immunohistochemical study was performed to detect the expression of β-catenin, MUC1 and c-met. Loss of membranous/cytoplasmic β-catenin expression in the tumour centre correlated with pT, loss at the invasion front with pTNM stage. MUC1 expression in the tumour centre correlated with lymph node metastasis and pTNM stage. c-Met did not show such associations. In multivariate survival analysis, loss of membranous/ cytoplasmic β-catenin expression as well as a strong MUC1 expression at the tumour invasion front represent independent predictors of a worse prognosis. On the other hand, c-met expression did not exhibit any prognostic value in this study.     

Diagnostic Significance of Two Fetal Proteins, {alpha}f and r{beta}s, in Hepatoma and Other Neoplastic Diseases

Incidences of f and rßs components were studied inserum samples from patients with various malignant and non-malignantdiseases. With the micro-Ouchterlony method, af was detectedexclusively in cases of hepatocellular carcinoma of which theincidence was 83.5%, whereas the incidence of rßswas 47.7%. However, rßs was also detected in somemalignant disease other than hepatocellular carcinoma, withan incidence of 16.5%, and was also detected in a small numberof cases of non-neoplastic liver diseases. Though not tumor-specific, rßs as one of the fetalserum proteins can be detected in cases of non-f-producing hepatomaand in cases of malignancies other than hepatoma. Thus, thedetection of this protein may be of diagnostic significance.Possible mechanism of the appearance of this protein in theblood was discussed.     

Enzymatic reduction of {beta}-ketonitrosamines

The reduction of N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-oxopropyl)propylamine(NOPPA) by hepatic and pancreatic cytosol and microsomes fromSyrian golden hamsters and Sprague-Dawley rats has been examined.All hepatic fractions reduced both substrates, although theactivity depended on the fraction tested and the cofactor employed(NADH or NADPH). Generally, hamster hepatic fractions containedhigher activity than the rat hepatic fractions and BOP was abetter substrate than NOPPA. Of the pancreatic fractions, onlycytosol exhibited reductase activity. The hamster cytosol wasable to utilise both cofactors, but the rat fraction exhibitedactivity only when NADPH was present. BOP was the better substratefor the pancreatic enzymes and in the presence of NADPH, therat and hamster activities were about equal. These results suggestthat the pancreatic reduction of BOP to HPOP is unlikely tobe a significant factor in the species-specific induction ofpancreatic cancer by BOP.     

Interrelationship between protein phosphatase 1 and TGF-{beta} in regulating motility and cytoskeletal architecture of endothelial cells

Motility of endothelial cells is a requirement for the vascularization of solid malignancies. While tumors have been shown to produce a host of angiogenic factors, including TGF-β, the mechanisms by which such factors regulate endothelial cell motility have not yet been defined. Thus, the role of the serine/threonine phosphatase PP-1 in regulating endothelial cell motility and cytoskeletal architecture was studied. The present study demonstrated that TGF-β stimulation of motility is dependent on PP-1. Likewise, TGF-β was shown to up-regulate paxillin expression through a process that was PP-1 dependent. The interplay between PP-1 and TGF-β was further observed by the induction of cell rounding and the loss of paxillin-actin co precipitations upon PP-1 inhibition and the compensation for these effects by TGF-β. Studies initiated to determine how PP-1 might regulate motility showed its role in maintaining cytoskeletal organization and its capacity to directly dephosphorylate the focal adhesion scaffolding protein paxillin. These studies suggest that the interplay between TGF-β and PP-1 regulates the motility of endothelial cells that is critical to the process of angiogenesis.     

Human tumor cell strains both unable to repair O6-methylguanine and hypersensitive to killing by human {alpha} and {beta} interferons

Human brain tumor cell strains were previously found by othersto be sensitive to growth inhibition by human inter-feron-ß(HuIFN-ß). We noticed that the sensitive strains weresome that we had found deficient in the repair of O⁶-methyl-guanine(O⁶MeG), a characteristic of 20% of the human tumor cell strainswe have studied. We confirmed this sensitivity to HuIFN-ß,and have further shown that human brain tumor cells which repairO⁶MeG are resistant to the growth inhibitory effects of HuIFN-ß.In addition, treatment with HuIFN- or HuIFN-ß resultedin more killing (reproductive inactivation) of six human tumorcell strains deficient in repairing O⁶-methylguanine in DNAthan did such treatment of six strains of cells proficient insuch repair. Further, we found two human lines, altered to becomeO⁶MeG repair deficient after establishment of the primary tumorcell culture, that were resistant to interferon. IFN treatmentproduced no DNA damage detectable by either chemical or biologicalassays. It is suggested that the genes responsible for resistanceto IFN treatment and to agents that produce O⁶MeG are oftencoordinately shut down.     

X-ray crystallographic analysis of ({+/-})-7{beta},8{alpha}-dihydroxy-9{beta},10{beta}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene: molecular structure of a 'syn' diol epoxide

X-ray crystallographic analysis has been used to define themolecular structure of the cis (syn) diol epoxide, (±)-7ß,8-dihydroxy-9ß,10ß-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.The two hydroxyl groups are oriented equatorially to the tetrahydrobenzenering, contrary to predictions and there is no intramolecularhydrogen bonding in the structure.     

Anti-cancer effects of morphine through inhibition of tumour necrosis factor-{alpha} release and mRNA expression

Morphine is mainly used to relieve pain in the terminal stageof cancer patients. We found that morphine has inhibitory effectson growth of various human cancer cell lines, with IC₅₀ from2.7 to 8.8 mM, and BALB/3T3 cells, with IC₅₀ of 1.5 mM. Althoughthe IC₅₀ values were relatively high, we decided to study themechanisms of anti-carcinogenic effects of morphine. Morphineinhibited activation of protein kinase C induced by teleocidin,one of the 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumourpromoters (IC₅₀, 1 mM). Based on our previous evidence thattumour necrosis factor-     

Phenobarbital promotes liver growth in c-myc/TGF-{alpha} transgenic mice by inducing hypertrophy and inhibiting apoptosis

Phenobarbital (PB) is a non-genotoxic liver tumor promoter usedextensively in initiation–promotion protocols. To determinethe mode of PB action, double transgenic mice overexpressingboth the c-myc and transforming growth factor (TGF)-     

Glutathione conjugation and DNA-binding of ({+/-})-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and ({+/-})-7{beta},8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in isolated rat hepatocytes

Isolated rat liver hepatocytes, previously depleted of glutathione(GSH) by treatment with diethylmaleate, were allowed to incorporate[³H]glycine into their GSH. Incubation of ³H-labelled cellswith ¹⁴C-labelled (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene((±)-BP-7,8-dihydrodiol) or (±)7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene(()-BPDE) revealed the formation of double labelled products.This together with evidence from amino acid analysis indicatesformation of GSH-conjugates of the highly carcinogenic BP-derivatives.Incubation of hepatocytes isolated from 3-methylcholanthrene(MC) treated rats with ³H-labelled (±)-BP-7,8-dihydrodiolor (±)-BPDE resulted in binding of radioactivity to DNA.Reduction of the intracellular level of GSH to 40% of the normallevel resulted in an approximate 2-fold increase in the DNA-bindingof either substrate. In addition there was a concurrent decreasein the amount of GSH-conjugates formed. These data clearly demonstratethat GSH participates in conjugation reactions with carcinogenic(±)-BP-7,8-dihydrodiol and (±)-BPDE and that theintracelluilar level of GSH is important in preventing reactiveintermediates from reacting with the DNA in intact cells.