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.     

Uptake and metabolism of {beta}-carotene and retinal by C3H/10T1/2 cells

Both ß-carotene (ß-C), a vitamin A precursor,and vitamin A itself have been shown to reversibly inhibit neoplastictransformation in 10T1/2 cells during the progression phaseof carcinogenesis. In order to determine whether the activityof ß-C in these cells may be attributed to conversionto vitamin A or is intrinsic to the carotenoid molecule, theuptake and metabolism of ß-C, and of retinal, theimmediate product of dioxygenase-cleavage of ß-C,was studied in 10T1/2 cells. Cellular uptake of 2.6 nmol/10⁶cells occurred 24h after treatment with 10^–5 M ß-C.Thereafter, cell levels remained relatively stable between 1and 2 nmol/10⁶ cells over the 1-week treatment period. Uponremoval of ß-C from the medium, cellular levels decreasedby {small tilde}80% in 2 weeks, then stabilized. Retinal wasrapidly and quantitatively converted to retinol by 10T1/2 cells,suggesting that the inhibitory action of retinal on neoplastictransformation in these cells is due to its conversion to retinol,and that any enzymatic conversion of ß-C to retinalby these cells would be expected to result in retinol as theend product. Using [¹⁴C]ß-C, we found no evidencefor formation of [¹⁴C]retinol, [¹⁴C]retinal or [¹⁴C]retinoicacid using sensitive HPLC. We therefore conclude that ß-Chas intrinsic chemopreventive activity in 10T1/2 cells, perhapsdue to its anti-oxidant properties.     

Inhibition of aflatoxin B1 -hepatocarcinogenesis in rats by {beta}-naphthoflavone

Effects of ß-naphthonflavoe (ßNF) on theactivity of hepatic microsomal aflatoxin B₁ (AFB₁)-4-hydroxylase- the cytochrome P-450-dependent enzyme system which catalyzesthe metabolism of AFB₁ to AFM₁ - and on AFB₁ induced in vivohepatocarcinogenesis were investigated in weanling male Fischerrats. A single i.p. injection of ßNF in doses of 20mg/kg and 150 mg/kg induced AFB₁ -4-hydroxylase 3- and 4-fold,respectively, 48 h post injection. Feeding of diet containing0.01% ßNF for a period of 9-weeks induced AFB₁ 2-fold.AFB₁, given by intubatlon in a dose of 25 µg five times/weekfor 8 weeks, produced 42 weeks later a 100% incidence of liverlesions (neoplastic foci, nodules or tumors), but feeding ßNFin diet at a con centration of 0.015% for one week prior toand during the 8 weeks of AFB treatment inhibited AFB₁ hepatocarcinogenesis by -7%. These results are in accord with the suggestionthat AFB₁ induction may be associated with the inhibition ofAFB₁ carcinogenesis, possibly occurring as a consequence ofaccelerated detoxi-fication of AFB₁ via its conversion to AFM₁     

Growth suppression of transformed BALB/c 3T3 cells by transforming growth factor {beta}1 occurs only in the presence of their normal counterparts

Transforming growth factor ß1 (TGF-ß1) enhancesthe yield of transformed foci of BALB/c 3T3 cells, but the continuouspresence of TGF-ß1 after foci formation inhibits thegrowth of transformed foci. The focus-forming ability of Ha-ras-,v-src-and PyMT-transformed cells growing on a monolayer of non-transformedcells was completely suppressed by TGF ß1, whereasgrowth of the transformed cells was little inhibited by TGF-ß1in the absence of their normal counterparts. The inhibitionby TGF-ß1 of focus formation by transformed BALB/c3T3 cells on a normal cell monolayer remained when TGF-ß1was removed from the culture medium after 2 weeks. However,the transformed cells were not killed, since they grew in cultureconditions under which only transformed cells are able to grow(soft agar). These results suggest that TGF-ß1 suppressesgrowth of transformed cells in the presence of normal cells.Furthermore, when non-transformed cells were treated with TGF-ß1before co-culture with Ha-ras-transformed cells, formation oftransformed foci was inhibited. When normal and transformedcells were cultured in the same dish but separated physically,focus formation was still Inhibited. On the other hand, TGF-ß1enhanced the growth and changed the morphology of non-transformedcells only in the presence of transformed counterparts. Thegrowth inhibitory effect of TGF-ß1 on transformedcells and its growth stlmulatory effect on non-transformed cellsin co-culture conditions suggest the induction of reciprocalparacrine growth regulatory factors. As TGF-ß1 inhibitsthe growth of transformed BALB/c 3T3 cells only in the presenceof their normal counterparts, a paracrine negative growth controlmechanism appears to be operating.     

NAMI-A inhibits the PMA-induced ODC gene expression in ECV304 cells: involvement of PKC/Raf/Mek/ERK signalling pathway

Imidazolium trans-imidazole dimethyl sulfoxide tetrachlororuthenate (NAMI-A) is a new compound active against lung metastasis of solid metastasizing tumours. While its in vivo effect has been studied, the molecular insights that underlie its action are largely unknown. Among the possible pathways responsible for malignant transformation, PKC arose as one of the most promising targets for new antineoplastic drugs. We demonstrated the capability of NAMI-A of inhibiting PMA induced-PKC activity in ECV304 in a dose-dependent fashion. Furthermore, NAMI-A through modulation of PKC activity has been proved capable of reducing the phorbol ester induced expression of ornithine decarboxilase (ODC) gene and to abrogate the activation of the Raf/MEK/ERK pathway. Taken together these results suggest that many of the in vivo outcomes of NAMI-A treatment may be the result of a direct action on PKC.     

Enhancement of genomic instability by 17{beta}estradiol in minisatellite sequences of X-ray-transformed mouse 10T1/2 cells

The female hormone 17ß-estradiol is involved in thedevelopment of breast cancer, an effect usually attributed toits capacity to stimulate the replication of preneoplastic andmalignant cells. In this study, we report that 17ß-estradiolenhances the onset of genomic rearrangements, a type of genomicinstability, in minisatellite sequences of malignant 10T1     

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.     

Cyr61 increases migration and MMP-13 expression via {alpha}v{beta}3 integrin, FAK, ERK and AP-1-dependent pathway in human chondrosarcoma cells

Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secretedand matrix-associated protein, which is involved in many cellularactivities such as growth and differentiation. However, theeffect of Cyr61 on migration activity in human chondrosarcomacells is mostly unknown. Here, we found that Cyr61 increasedthe migration and expression of matrix metalloproteinase (MMP)-13in human chondrosarcoma cells (JJ012 cells). RGD peptide, vβ3monoclonal antibody and mitogen-activated protein kinase (MEK)inhibitors (PD98059 and U0126) but not RAD peptide inhibitedthe Cyr61-induced increase of the migration and MMP-13 upregulationof chondrosarcoma cells. Cyr61 stimulation increased the phosphorylationof focal adhesion kinase (FAK) and extracellular signal-regulatedkinase (ERK). In addition, activator protein-1 (AP-1) decoyoligodeoxynucleotide also suppressed the MMP-13 messenger RNAand enzyme activity enhanced by Cyr61. Moreover, Cyr61 increasedthe binding of c-Fos and c-Jun to the AP-1 element on the MMP-13promoter. Taken together, our results indicated that Cyr61 enhancesthe migration of chondrosarcoma cells by increasing MMP-13 expressionthrough the vβ3 integrin receptor, FAK, ERK, c-Fos/c-Junand AP-1 signal transduction pathway. Abbreviations: AP-1, activator protein-1; AS, antisense; Cyr61, cysteine-rich 61; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; mAb, monoclonal antibody; MEK, MAPK kinase; MMP, matrix metalloproteinase; mRNA, messenger RNA; MS, missense; NF-B, nuclear factor-kappa B; ODN, oligonucleotide; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; qPCR, quantitative real-time polymerase chain reaction; siRNA, small-interfering RNA     

Inhibition of {beta}-adrenergic response in cultured epidermal cells by phorbol myristate acetate

Treatment of cultured epidermal cells with the tumor promoter,phorbol myristate acetate (PMA), results in a 75% in hibitionof the normal isoproterenol stimulated 7-fold in crease in cAMPaccumulation. Maximum inhibition occurred 3 h after PMA treatment.The effect of phorbol ester on the ß-adrenergic systemin mouse epidermis thus appears to be due to its action on theepidermal cell membrane and does not require mediation by macrophages.     

Effect of three tumour promoters on the stability of hepatocyte cultures and apoptosis after transforming growth factor-{beta}1

Tumour promoters like the anti-androgen cyproterone acetate(CPA), the peroxisome proliferator nafenopin (NAF) and phenobarbital(PB) stimulate liver growth in rodents. Transforming growthfactor-ß1(TGF-ß1) is expressed in liversafter treatment with CPA (Oberhammer et at., submitted) andsome peroxisome proliferators. In this paper we describe theinfluence of CPA, NAF and PB on the stability of hepatocytecultures and induction of apoptosis by TGF-ß1. Allthree tumour promoters had a stabilizing effect on confluentmonolayers of hepatocytes, partially preventing the usuallyoccurring dedifferentiation and detachment processes. CPA onits own was able to induce apoptosis at the high dose of 10µM. No induction of apoptosis could be observed afterPB and NAF. At any dose above 0.01 µM CPA enhanced TGF-ß1-inducedapoptosis (5.8-fold increase with 10 µM CPA). Thus thecombination of 10 µM CPA and 1 ng/ml TGF-ß1induced apoptosis in 90% of the plated hepatocytes. At a highdose (10 µM) NAF produced a 35% reduction in apoptosisinduced by TGF-ß1 in parallel with a stabilizing effecton cell number. PB did not affect the rate of apoptosis inducedby TGF-ß1. As demonstrated by immunohistochemicaldetection of PCNA, TGF-ß1 prevented induction of PCNAby epidermal growth factor (EGF). No induction of PCNA was observablein CPA-treated cultures. In untreated and EGF-treated culturesTGF-ß1 was able to induce apoptosis to the same extentwithin 30 h. In CPA-treated cultures this period was shortenedto 12 h. Thus CPA shortens the lag phase of induction of apoptosisby shifting hepatocytes to a point before S phase, where theyare highly susceptible to TGF-ß1-induced apoptosis.     

Effect of some peroxisome proliferators on transforming growth factor-{beta}1 gene expression and insulin-like growth factor n/mannose-6-phosphate receptor gene expression in rat liver

Male Sprague-Dawley rats were given daily oral doses of eithercorn oil (control), 80 mg/kg nafenopin (NAF), 50 mg/kg methylclofenapate(MCP), 50 mg/kg Wy-14, 643 (WY) or 250 mg/kg clofibric acid(CA) for 7 days. All four compounds increased relative liverweight and produced hepatic peroxisome proliferation as assessedby induction of both peroxisomal (palmitoyl-CoA oxidation) andmicrosomal (lauric acid 12-hydroxylase) fatty acid oxidisingenzyme activities. RNA was extracted from liver samples andanalysed for expression of transforming growth factor-ß₁(TGF-ß₁) and the insulin-like growth factor II/mannose-6-phosphate(IGFII/Man6P) receptor (which may be involved in transportinglatent TGF-ß₁, into hepatocytes). TGF-ß₁mRNA levels were increased to 151 –178% of control byall four compounds, whereas NAF, MCP and WY, but not CA, increasedIGFII/Man6P receptor mRNA levels to 195–209% of control.The induction of TGF-ß₁ and IGFII/Man6P receptor expressionby short term treatment with peroxisome proliferators may representan adaptive response to limit the initial hyperplastic effectsof such compounds.     

{beta}-Carotene and canthaxanthin inhibit chemically- and physically- induced neoplastic transformation in 10T1/2 cells

We have studied the effects of ß-carotene(ß03-C),a vitamin A precursor of plant origin, and canthaxanthin (CTX),a non-provitamin A carotenoid, on the neoplastic transformationof C3H/10T1/2 murine fibroblast cells. Chemical transformationin this well-characterized cell system has previously been shownto be reversibly inhibited by retinoids, compounds with vitaminA-like activity. Here we show that both ß-C and CTXinhibit 3-methycholanthrene (MCA)-induced transformation withED⁵⁰S of 9 x10-⁷M and 2x10-⁷ M, respectively. Both carotenoidsfailed to inhibit X-ray-induced transformation when the cellswere treated prior to and during irradiation. However, whenthe drugs were added 1 week after X-irradiation and maintainedin the medium thereafter, as in the chemical transformationprotocol, both carotenoids inhibited subsequent developmentof transformed foci in a dose-dependent manner. Again, CTX wasmore effective than ß3-C, such that 3 x 10-⁶M completelyinhibited radio-genicaly-induced foci. Similar to the previouslydescribed action of retinoids, the inhibition of MCA-inducedtransformation was reversible; developed upon removal of thedrug, transformed foci developed within 2 weeks, indicatingthat the carotenoids were not specifically toxic to initiatedcells. Although both carotenoids caused a small dose-dependentdecrease in the growth rate of both parental and initiated 10T1/2cells, they did not markedly affect colony size or number whenthe cells were treated as in the transformation assays, nordid they influence the expression of neoplasia of two transformedcell lines. Although the actions of ß3-C and CTX aresimilar to those of retinoids in the 10T1/2 system, we suggestthat the carotenoids act via a different mechanism, since CTXcannot be converted to active retinoids in mammalian cells,and there is no evidence that 10T1/2 cells can convert ß-Cto vitamin A. We suggest that the carotenoids, lipid anti-oxidantproperties may be responsible for their inhibitory actions ontransformation.     

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.     

Macrophages promote the invasion of breast carcinoma cells via a colony-stimulating factor-1/epidermal growth factor paracrine loop

Previous studies have shown that macrophages and tumor cells are comigratory in mammary tumors and that these cell types are mutually dependent for invasion. Here we show that macrophages and tumor cells are necessary and sufficient for comigration and invasion into collagen I and that this process involves a paracrine loop. Macrophages express epidermal growth factor (EGF), which promotes the formation of elongated protrusions and cell invasion by carcinoma cells. Colony stimulating factor 1 (CSF-1) produced by carcinoma cells promotes the expression of EGF by macrophages. In addition, EGF promotes the expression of CSF-1 by carcinoma cells thereby generating a positive feedback loop. Disruption of this loop by blockade of either EGF receptor or CSF-1 receptor signaling is sufficient to inhibit both macrophage and tumor cell migration and invasion.     

Differential formation of beta-catenin/lymphoid enhancer factor-1 DNA binding complex induced by nitric oxide in mouse colonic epithelial cells differing in adenomatous polyposis coli (Apc) genotype

Increased cytoplasmic beta-catenin levels and the associated nuclear beta-catenin/T-cell factor (Tcf)-lymphoid enhancer factor (LEF) complex formation have been frequently found in colon cancer. In this context, overproduction of nitric oxide (NO) attributable to inflammatory stimuli in diseases such as ulcerative colitis and Crohn's disease may-contribute to colonic carcinogenesis. Therefore, we examined the modulation by NO of cytoplasmic beta-catenin levels and the formation of the nuclear beta-catenin/LEF-1 DNA binding complex in conditionally immortalized mouse colonic epithelial cells that differed in adenomatous polyposis coli (Apc) genotype, namely young adult mouse colon (YAMC; Apc+/+) and immortal mouse colon epithelium (IMCE; ApcMin/+). Unlike most colon cancer cell lines, this pair of cell lines has either nondetectable or low basal level of beta-catenin when they are cultured under nonpermissive and nonproliferative conditions. Using electrophoretic mobility shift assays, we found that NO-releasing agents (E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide and S-nitroso-N-acetylpenicillamine greatly enhanced the formation of beta-catenin/LEF-1 DNA binding complex in a concentration- and time-dependent fashion in YAMC and IMCE cells. Significantly, IMCE cells showed a markedly greater amount of nuclear beta-catenin/LEF-1 DNA binding complex in response to NO. Super shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Western blot analysis of the soluble cytoplasmic fractions demonstrated that these NO donors caused differential accumulation of cytoplasmic beta-catenin in YAMC and IMCE. In conclusion, this study indicates that the defective beta-catenin degradation machinery attributable to ApcMin/+ mutation in IMCE cells not only affects basal levels but also contributes to NO-induced dysregulation of cytoplasmic beta-catenin and nuclear beta-catenin/LEF-1 DNA binding complex formation.