Okadaic Acid and Phorbol Esters: Comparative Effects of These Tumor Promoters on Cell Transformation, Intercellular Communication and Differentiation in vitro

Okadaic acid is a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoter in the mouse skin carcinogenesis system. Here we report on the in vitro activity of okadaic acid in 3 assay systems: BALB/c 3T3 cell transformation, gap junctional intercellular communication (GJIC) in various cell types, and inhibition of induction of differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells. The activity of okadaic acid was compared to that of the phorbol ester tumor promoters TPA and phorbol-12,13-didecanoate (PDD). In a test system involving a 2-week exposure of BALB/c 3T3 cells following 3-methylcholanthrene initiation, okadaic acid at a concentration of 10 ng/ml was equipotent to PDD as a promoter of cell transformation (4.9 and 3.7 foci/dish, respectively). Longer exposures to okadaic acid resulted in cytotoxicity. Okadaic acid-generated as well as PDD-generated transformed foci displayed a selective lack of GJIC between focus cells and surrounding normal cells, i.e., transformed cells communicate among themselves but not with surrounding cells. However, in contrast to TPA, there was no inhibition by okadaic acid, except at toxic doses, of homologous GJIC in BALB/c 3T3 cells or human and mouse keratinocytes. Furthermore, okadaic acid, unlike TPA, did not inhibit MEL cell differentiation. Together, these results indicate that okadaic acid acts as a promoter of cell transformation but that its mechanism of action is different from that of the phorbol ester tumor promoters.     

Promotion of BALB/3T3 Cell Transformation by the Okadaic Acid Class of Tumor Promoters, Okadaic Acid and Dinophysistoxin-1

Okadaic acid and dinophysistoxin-1 are non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoters, which enhance chemically induced tumorigenesis on mouse skin through a different mechanism from that of TPA. In the present study, we examined the promoting effects of these okadaic acid class tumor promoters on a two-stage transformation using BALB/3T3 cells which was designed to simulate in vivo two-stage carcinogenesis. Cells were treated first with a low dose of the initiator 3-methylcholanthrene (MCA) and then with a test chemical. Okadaic acid and dinophysistoxin-1 significantly enhanced the MCA-induced cell transformation. Okadaic acid tetramethyl ether, an inactive compound, did not affect the transformation of MCA-treated cells. The okadaic acid class of tumor promoters failed to induce transformation without pretreatment by MCA. Okadaic acid did not show initiating activity in the two-stage transformation assay in which cells were treated first with okadaic acid and then with TPA. These results indicate that this transformation assay with BALB/ 3T3 cells is useful to predict tumor-promoting activity of non-TPA-type as well as TPA-type tumor promoters, before long-term in vivo two-stage carcinogenesis experiments are carried out.     

Promotion of BALB/3T3 cell transformation by the okadaic acid class of tumor promoters, okadaic acid and dinophysistoxin-1

Okadaic acid and dinophysistoxin-1 are non-12-O-tetradecanoylphorbol-13-acetate (non-TPA)-type tumor promoters, which enhance chemically induced tumorigenesis on mouse skin through a different mechanism from that of TPA. In the present study, we examined the promoting effects of these okadaic acid class tumor promoters on a two-stage transformation using BALB/3T3 cells which was designed to simulate in vivo two-stage carcinogenesis. Cells were treated first with a low dose of the initiator 3-methylcholanthrene (MCA) and then with a test chemical. Okadaic acid and dinophysistoxin-1 significantly enhanced the MCA-induced cell transformation. Okadaic acid tetramethyl ether, an inactive compound, did not affect the transformation of MCA-treated cells. The okadaic acid class of tumor promoters failed to induce transformation without pretreatment by MCA. Okadaic acid did not show initiating activity in the two-stage transformation assay in which cells were treated first with okadaic acid and then with TPA. These results indicate that this transformation assay with BALB/3T3 cells is useful to predict tumor-promoting activity of non-TPA-type as well as TPA-type tumor promoters, before long-term in vivo two-stage carcinogenesis experiments are carried out.     

Transforming growth factor beta as a potent promoter in two-stage BALB/c 3T3 cell transformation

We have tested transforming growth factor beta (TGF beta) in the two-stage BALB/c 3T3 cell transformation assay for possible tumor-promoting activity, since it has several effects similar to those of tumor-promoting phorbol esters. After initiation of BALB/c 3T3 cells with 3-methylchol-anthrene, treatment with TGF beta at 1 ng/ml alone or in combination with epidermal growth factor (EGF) for 4 weeks enhanced the number of transformed foci by 5- to 6-fold in comparison with uninitiated cells. Initiation treatment alone induced no or very few transformed foci in several assays. Treatment with phorbol-12,13-didecanoate (PDD) at 100 ng/ml for 4 weeks enhanced the number of transformed foci in initiated BALB/c 3T3 cells by 4- to 5-fold in comparison with uninitiated cells. Thus, TGF beta at 1 ng/ml is as potent as PDD at 100 ng/ml for tumor-promoting activity in the two-stage BALB/c 3T3 cell transformation assay. The enhancing effect of TGF beta was dose-related in the dose range tested (0.03-1 ng/ml) and was not reversible. Some of the foci induced by combined MCA-TGF beta-EGF treatment were cloned, and eight out of nine clones tested produced tumors in nude mice. TGF beta (1 ng/ml) plus EGF (2 ng/ml) increased the saturation density to a similar extent as PDD (100 ng/ml) but did not affect the growth of BALB/c 3T3 cells. We observed no change in junctional intercellular communication, as measured by the dye transfer method, when cells were treated with TGF beta during the two-stage BALB/c 3T3 cell transformation assay. Nevertheless, there was selective communication between transformed and surrounding nontransformed cells; MCA-TGF beta transformed cells intercommunicated among themselves but not with surrounding nontransformed cells. Our results indicate that TGF beta has potent tumor-promoting activity in vitro, but that this activity is not mediated by a complete blockage of intercellular communication, as is suggested for phorbol ester tumor promoters.     

Inhibition of tumor promoter activity toward mouse fibroblasts and their in vitro transformation by tissue inhibitor of metalloproteinases- 1 (TIMP-1)

Tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural inhibitor of matrix metalloproteinases (MMPs), is known to inhibit invasion and metastasis of tumor cells. In the present study we examined anti-tumor promoter activity of TIMP-1 and its effect on in vitro cell transformation using BALB/3T3 cells in low serum culture medium. In the dye transfer assay the tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) continuously blocked gap-junctional intercellular communication (GJIC) of BALB/3T3 cells in confluent phase. TIMP-1 did not prevent transient inhibition of GJIC induced by TPA, but it quickly restored the reduced GJIC level to the control level. The recovery of GJIC was dependent on the concentration of TIMP-1 from 1 to 1000 ng/ml. In an in vitro two-stage transformation assay in which BALB/3T3 cells were treated with 0.5 microg/ml N-metyl-N'-nitro-N-nitrosoguanidine as initiator and 100 ng/ml TPA as promoter, TIMP-1 at concentrations > 10 ng/ml inhibited the focus formation of transformed cells by approximately 60%. TIMP-2 and a synthetic MMP inhibitor showed a similar inhibitory activity on in vitro cell transformation. Furthermore, zymographyic analysis showed that TPA treatment of BALB/3T3 cells induced secretion of gelatinase B and stromelysin-1 into the culture medium. These results indicate that TIMP-1 and TIMP-2 have inhibitory activity on in vitro transformation of cells. It seems likely that TPA-inducible MMPs are involved in carcinogenesis and TIMPs have a protective role against carcinogenesis in vivo.      

A new concept of tumor promotion by tumor necrosis factor-alpha, and cancer preventive agents (-)-epigallocatechin gallate and green tea--a review

The study of tumor promotion in rodent carcinogenesis using chemical tumor promoters has revealed various tumor promotion pathways, such as the 12-O-tetradecanoylphorbol-13-acetate (TPA) pathway mediated through activation of protein kinase C, and the okadaic acid pathway mediated through inhibition of protein phosphatases 1 and 2A (PP-1 and PP-2A). We previously demonstrated that application of TPA and okadaic acid induced tumor necrosis factor-alpha (TNF-alpha) gene expression in mouse skin, but that tautomycin, which is an inhibitor of PP-1 and PP-2A and not a tumor promoter on mouse skin, did not. Moreover, we found that TNF-alpha stimulated transformation of BALB/3T3 cells initiated with 3-methylcholanthrene 1,000 times stronger than did TPA (Cancer Res. 53, 1982-1985, 1993). This evidence demonstrates a link between the okadaic acid pathway and the endogenous tumor promotion pathway of TNF-alpha. Recently we presented the first evidence that tumor promotion in TNF-alpha(-/-) mice was significantly depressed compared with TNF-alpha(+/+) mice. Thus, in human carcinogenesis, we think that TNF-alpha and other inflammatory cytokines in preneoplastic lesion stimulate tumor promotion and progression of initiated cells as well as premalignant cells. The first part of this paper reports on this TNF-alpha tumor promotion pathway. In the second part, we report a promising screening method for cancer preventive agents, based on evidence that pretreatment with agents such as tamoxifen, sulindac, 1alpha, 25-(OH)2 vitamin D3, quercetin, caffeic acid phenethyl ester, and (-)-epigallocatechin gallate (EGCG) commonly inhibited TNF-alpha release from BALB/3T3 cells induced by okadaic acid. EGCG, the main constituent of Japanese green tea, and green tea itself are acknowledged cancer preventives in Japan, and this paper presents evidence of their effectiveness in both a high-risk group and the general population.     

Orthovanadate, an inhibitor of protein tyrosine phosphatases, acts more potently as a promoter than as an initiator in the BALB/3T3 cell transformation

The phosphorylation and dephosphorylation of proteins are critical in cellular signal transduction. Phorbol esters and okadaic acid, which affect protein phosphorylation, are potent promoters in mouse skin carcinogenesis and cell transformation in vitro. Orthovanadate inhibits protein tyrosine phosphatases and causes hyperphosphorylation of cellular proteins. We have performed two-stage transformation assays using BALB/3T3 cells to determine the major activity of orthovanadate (1-10 microM) for transformation. This chemical acted as a weak initiator because its initiating treatment produced a significant, though small, number of transformed foci in the presence of promoting treatment by 12-O-tetradecanoylphorbol-13-acetate (TPA) but not in the absence of TPA. Promoting treatment by orthovanadate markedly enhanced the transformation of the cells pretreated by a subthreshold dose of 3- methylcholanthrene (MCA) but not of non-pretreated cells. Superiority of promoting over initiating activity of orthovanadate was confirmed by an assay carried out in the reversed treatment sequence (orthovanadate and then MCA), where the transformation frequency was conspicuously decreased compared with the regular treatment sequence. The transformed foci in the cultures treated by orthovanadate, following MCA treatment, continued to grow in normal medium, showing cell proliferation independent of orthovanadate. Orthovanadate, in addition to TPA and okadaic acid, will be a useful reagent for studying the signaling cascades responsible for tumor promotion.      

Association of viral oncogene-induced changes in gap junctional intercellular communication and morphological transformation in BALB/c3T3 cells

In order to study the relationship between altered gap junctionalintercellular communication (GJIC) and induction of cell transformationby oncogenes, we transfected six viral oncogenes into BALB/c3T3A31-1-1 cells. BALB/c3T3 cells with v-src, v-ras or polyomamiddle T (PyMT) genes grew in soft agar and formed distincttransformed foci in the absence or presence of a vast excessof non-transfected cells. On the other hand, those with v-myc,v-fos or polyoma large T (PyLT) genes expressed less distinctlytransformed phenotypes (less transformed morphology, highersaturation density than non-transfected counterparts and lessgrowth in soft agar), and did not form distinct foci in coculturewith non-transformed cells. When their homologous GJIC capacitieswere examined by the microinjection/dye transfer assay, no decreasein GJIC was observed in any of the v-onc-transformed cells.Non-transformed and all v-onc-transformed cell lines expressedsimilar levels of connexin 43 mRNA. v-myc-, v-fos- and PyLT-transformedcells, but not v-ras-, v-src- and PyMT-transformed cells wereable to communicate heterologously with non-transformed cells.Tumor promoting phorbol esters strongly inhibited GJIC of non-transformedand all v-onc-transformed BALB/c3T3 cell lines. In coculturesof v-myc-, v-fos- or PyLT-transformed cells with non-transformedBALB/c3T3 A31-1-1 cells, 12-O-tetradecanoylphorbol-13-acetate(TPA) increased the number of transformed foci. However, whenthese v-onc-transformed cells were co-cultured with non-transformedBALB/c3T3 A31-1-13 cells (which lose GJIC at growth confluence,as if TPA had been added), no morphologically transformed fociappeared. These results suggest that factors other than GJICare involved in the suppression of oncogene-transformed cellsby surrounding normal counterparts.     

Okadaic acid: a reversible inhibitor of neoplastic transformation of mouse fibroblasts

Okadaic acid (OA) is a potent inhibitor of serine/threonine-specific protein phosphatases types 1 and 2A at nanomolar concentrations in cell-free assays and has tumor promoting activity in vivo. We have found that at non-toxic, nanomolar concentrations, OA concentration dependently inhibits the induction of focus-forming transformed cells by the "complete" and "two-stage" protocols in the C3H/10T1/2 mouse fibroblast transformation assay. This inhibitory effect was fully reversible upon removal of OA from the culture medium of carcinogen-treated cells, indicating that OA was not selectively toxic to initiated or transformed cells. Additional treatment with the phorbol ester tumor promoter, TPA, was required to promote the induction of transformed cells after the removal of OA in the two-stage transformation assay. At concentrations that inhibited neoplastic transformation, OA inhibited a type 2A-like phosphohistone protein phosphatase in homogenates of C3H/10T1/2 cells. It is postulated that OA inhibited an early protein phosphatase-sensitive event in the process of in vitro neoplastic transformation by C3H/10T1/2 fibroblasts and had the effect of maintaining carcinogen-treated cells in an initiated state.     

Anti-transforming nature of ascorbic acid and its derivatives examined by two-stage cell transformation using BALB/c 3T3 cells

The anti-transforming effects of sodium ascorbate and its stable derivatives were examined in the two-stage transformation assay. When BALB/c 3T3 cells were treated with 0.2 microg/ml 20-methylcholanthrene as an initiator, and 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate as a promoter, the addition at the promotion stage of L-ascorbic acid-2-phosphate ester magnesium (APM) was most marked in the inhibition of transformation. The inhibitory effects of sodium ascorbate and ascorbic acid-2-glucoside (AG) were comparable, but weaker than those of APM; L (+)-ascorbic acid-2-sulfate ester disodium 2H(2)O showed little effect. When phorbol 12, 13-didecanoate or tumor necrosis factor alpha (TNF-alpha) were used as promoters, APM also effectively suppressed transformation.     

Role of junctional intercellular communication in hepatic carcinogenesis in rats

Two major alterations in gap junctional intercellular communication (GJIC) during experimental carcinogenesis have been seen: a decrease of its capacity during tumor promotion and a selective loss of intercellular communication between transformed cells and surrounding normal counterparts. These data, first discovered in our laboratory on mouse fibroblasts (BALB/c 3T3), were partly confirmed in rat liver epithelial cells. In such cells, the decreased GJIC was related to their level of transformation. Tissue-specific effect of tumor promoters is also seen in the inhibition of GJIC; administration of phenobarbital decreased the level of GJ mRNA in the liver, but not in other organs tested. Phenobarbital also inhibited GJIC among adult rat hepatocytes co-cultured with BALB/c 3T3 cells but not among the mouse fibroblasts. Moreover, our findings on the diminished level of GJ mRNA in tumors of rat liver support the importance of acquired selective GJIC in rat liver carcinogenesis.     

Specific viral oncogenes cause differential effects on cell-to-cell communication, relevant to the suppression of the transformed phenotype by normal cells

We have studied growth regulation in mixed cultures of normal and oncogene-transformed 3T3 cells. The NIH 3T3 cells transformed by myc, src, and ras showed comparable cloning efficiency in semisolid medium. However, when they were plated on plastic with an excess of normal mouse embryo fibroblasts, BALB/c 3T3 ClA31-1-1, ras- and src-transformed cells were able to form distinct foci on the layer of density-arrested normal cells, whereas myc-transformed cells lacked this ability. In order to determine whether suppression or expression of the transformed phenotype could be correlated with the ability of the different cell populations to communicate, gap-junctional intercellular communication (IC) was measured by the Lucifer yellow dye transfer assay in coculture of normal and transformed cells. The dye was observed to spread from BALB/c 3T3 to myc-NIH 3T3 cells, indicating the presence of IC between these two cell types. In contrast no passage of Lucifer yellow was observed between src-NIH 3T3 or ras-NIH 3T3 and BALB/c 3T3. Addition of a phorbol ester tumor promoter, phorbol-12,13-didecanoate, efficiently rescued proliferation and focus formation by myc-transformed cells. The tumor promoter was able to inhibit IC in BALB/c 3T3 cells, although this response greatly varied among the different oncogene transformed clones. Tumorigenicity in nude mice strongly correlated with growth behavior in vitro: myc-transformed cells were either nontumorigenic or slowly tumorigenic, and src- and ras-transformed cells were highly tumorigenic. These data suggest an important role of IC in modulating abnormal growth behavior in vitro and in vivo.     

Phorbol ester-induced adhesion of murine erythroleukemia cells: possible involvement of cellular proteases

Phorbol ester tumor promoters produce a rapid increase in adhesivenessof murine erythroleukemia (MEL) cells. Following treatment with12-O-tetradecanoyl-phorbol-13-acetate (TPA) and other tumorpromoters, these cells adhere to the surface of the culturedish or become agglutinated to each other. Structurally relatedcompounds which are devoid of tumor promoting activity failedto induce agglutination of MEL cells. Pentamidine isethionate(PI) and tosylamide-phenylethyl-chloromethyl ketone, two knowninhibitors of trypsin-like enzymes, prevent the phorbol esters-inducedadherence and agglutination. A short exposure to TPA resultsin an increase in protease activity at the alkaline pH range.This TPA-induced proteolytic activity is inhibited by PI. Inductionof erythroid differentiation by hexamethylene-bisacetamide isassociated with a decrease in TPA-induced cell adhesion andTPA-induced proteolytic activity. Taken together, these resultssuggest the participation of an alkaline proteolytic activityin the membranal changes evoked by phorbol esters.     

Okadaic acid inhibits PDGF-induced proliferation and decreases PDGF receptor number in C3H/10T1/2 mouse fibroblasts.

Okadaic acid is both a potent inhibitor of protein serine/threonine phosphatases and a tumor promoter in the mouse skin model. We have previously shown that at non-toxic nanomolar concentrations okadaic acid reversibly inhibits induction (promotion) by PDGF of transformed cells by the 'complete' and 'two-stage' protocols in the C3H/10T1/2 mouse fibroblast transformation assay. In the present study we have demonstrated that treatment of confluent and proliferatively quiescent C3H/10T1/2 mouse fibroblasts with low doses of okadaic acid inhibits the platelet-derived growth factor (PDGF)-induced mitogenic response. This inhibition is accompanied by a loss of PDGF binding sites, a decreased PDGF-induced phosphatidylinositol turnover and a decrease in the PDGF-induced intracellular calcium signal. The decrease in the PDGF-generated intracellular signalling processes represents a mechanism by which okadaic acid inhibits PDGF-induced proliferation and the promotion of in vitro neoplastic transformation by PDGF.     

Identification and quantification of a carcinogen-induced molecular initiation event in cell transformation.

All transformed foci of Balb/c 3T3 clone A31-1-1 cells induced by 7,12-dimethylbenz[a]anthracene (DMBA) (42 out of 42 examined) contained an A to T transversion at codon 61 (A182 to T) of the Ki-ras gene. The transformants induced by other carcinogens tested did not contain such a mutation, except one out of nine 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced transformed foci. Thus, we hypothesized that this mutation is a specific DMBA-induced initiating event in Balb/c 3T3 cell transformation and we have measured its frequency of induction before transformation occurs, employing our recently developed method. Such mutations can be detected in the cell population as early as 3 days after exposure to DMBA. The same mutation was also detected in the Ha-ras gene. No detectable level (< 10(-6) of these mutations was induced by other carcinogens tested. The mutation frequency of the Ha-ras gene reached a plateau after 1 week's exposure, but that of the Ki-ras gene continued to increase. These results suggest that the A182 to T mutation of the Ki-ras gene, but not that of the Ha-ras gene, contributes to morphological transformation of Balb/c 3T3 cells. We have demonstrated that the level of expression of ras genes determines the rate of recruitment of cells into transformation. Quantitative analysis of the frequencies of ras gene mutations (initiation) and of transformation suggests that about 25% of those cells with the Ki-ras mutation were recruited into the full transformation process and that, in the presence of the tumor promoter TPA, about 56% of them completed morphological cell transformation.     

Comparative effects of a complete tumor promoter, TPA, and a second-stage tumor promoter, RPA, on intercellular communication, cell differentiation and cell transformation

The biological activities in vitro of the incomplete (second-stage)tumor promoter, 12-O-retinoyl phorbol-13-acetate (RPA), andthe complete tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate(TPA) were compared. The doses of TPA and RPA necessary to inhibitthe specific binding of [³H]-phorbol-12,13-dibutyrate ([³H]PDBu)to BALB/c 3T3 cells (50% inhibition doses; ID₅₀; 8–13ng/ml) were very similar; however, RPA was less potent thanTPA in inhibiting [³H]-PDBu binding to Friend erythroleukemiacells (FELC). Intercellular communication between BALB/c 3T3cells, measured by transfer of microinjected fluorescent dye(Lucifer Yellow), was inhibited by RPA as well as by TPA; TPAwas about five times more potent than RPA. RPA also inhibitedFELC differentiation induced by hexamethylene bisacetamide (HMBA)but not the differentiation of a TPA-resistant clone. The dose-responsesof these two compounds in inhibiting differentiation of bothTPA-sensitive and resistant FELC were very similar. When TPAand RPA were compared in their promoting activity of in vitrocell transformation of BALB/c 3T3 cells initiated with 3-methykholanthrene(MCA, 0.1 µg/ml), both TPA and RPA significantly increasedthe yield of morphologically transformed foci, and RPA was 10times more potent than TPA. These results suggest that RPA andTPA share many common in vitro biological effects and that thesein vitro studies do not allow us to delineate clearly the effectof a second-stage tumor promoter from that of complete tumorpromoters such as TPA.     

The induction of epidermal transglutaminase and terminal differentiation by tumor promoters in cultured epidermal cells

Previous studies had indicated that complete skin tumor promoters of the phorbol ester class induce epidermal transglutaminase and cornification in a subpopulation of cultured mouse epidermal basal cells in proportion to their promoting properties. This report describes the effect of promoting agents other than phorbol esters on the differentiation response and explores the pharmacological basis for the heterogeneity of responsiveness among subpopulations. The potent indole alkaloid skin tumor promoter, teleocidin, induces transglutaminase to the same extent or greater than 12-O-tetradecanoylphorbol-13-acetate (TPA). The highly inflammatory and cytotoxic non-promoting agent resiniferotoxin is not an inducer of transglutaminase. Incomplete skin tumor promoters, mezerein and retinyl phorbol acetate, were as potent as TPA as inducers of transglutaminase. Anthralin and benzoyl peroxide, skin tumor promoters which do not bind to the phorbol ester receptor, do not induce transglutaminase. TPA was used to study the influence of the state of epidermal maturation at the time of exposure on the differentiation response. Epidermal basal cells were induced to differentiate by elevating extracellular calcium to 1.2 mM. TPA markedly accelerates the differentiation program when given simultaneous with exposure to 1.2 mM Ca2+ as indicated by measurements of DNA synthesis, transglutaminase activity and cornified cells. Furthermore, epidermal cells committed to differentiate by switching to 1.2 mM Ca2+ medium remain responsive to the differentiative effects of TPA for at least 5 h. These results indicate that the induction of transglutaminase activity and cornification in epidermal basal cells is characteristic of phorbol ester promoters or other agents that bind to the phorbol ester receptor but is not characteristic of all skin tumor promoters. This result suggests that the phorbol ester receptor regulates epidermal differentiation. The state of differentiation of epidermal cells at the time of phorbol ester exposure may determine whether the cellular response will be in a proliferative or differentiative pathway.     

Cross-resistance to tumour promoters in human cancer cell lines resistant to adriamycin or cisplatin

The growth inhibitory effect of tumour promoters on human leukaemia and lung cancer cell lines was examined using the [3-(4,5 dimethylthiazol)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. The four cell lines used were the K562 human leukaemia cell line, its adriamycin (ADM)-resistant subline (K562/ADM), which shows the mdr phenotype, PC-9 (a human lung adenocarcinoma cell line) and its cisplatin (CDDP)-resistant subline (PC-9/CDDP), which does not show the mdr phenotype. Phorbol 12-tetradecanoate-13-acetate (TPA) and the TPA-type tumour promoters, aplysiatoxin and debromoaplysiatoxin, inhibited the growth of the two parental cell lines, K562 and PC-9. The non-TPA-type tumour promoter, okadaic acid, also inhibited the growth of the two parental cell lines in a dose-dependent manner. TPA-type and okadaic acid inhibited the growth of K562/ADM more weakly than that of K562, and showed no growth inhibition in PC-9/CDDP. Anhydrodebromoaplysiatoxin, an inactive derivative of the TPA-type tumour promoter, could suppress the growth of K562 and K562/ADM only at high concentration (more than 50 pM) and it showed similar growth inhibitory effects on the two cell lines. Okadaic acid tetramethyl ether, the inactive form of the non-TPA-type tumour promoter did not inhibit the growth of any of the cell lines. The growth inhibitory effect of these compounds was well correlated with their tumour-promoting activity. A study of the accumulation of okadaic acid revealed that the amount of 3H-okadaic acid in K562/ADM and PC-9/CDDP was similar to that in their parental cells indicating that cross-resistance to this tumour promoter in the drug-resistant cell lines is not due to a difference in the amount of drug accumulated in sensitive and resistant cells. These results suggest the presence of another common mechanism for resistance to ADM and CDDP as well as to TPA- or non-TPA-type tumour promoters.     

Discrete roles of cytokines, TNF-alpha, IL-1, IL-6 in tumor promotion and cell transformation.

Based on our previous results, which pointed to tumor necrosis factor-alpha (TNF-alpha) as the essential cytokine in tumor promotion in mouse skin, we present here three principal findings related to the specific roles of TNF-alpha, interleukin-1 (IL-1) and IL-6 in tumor promotion (using TNF-alpha- and IL-6-deficient mice) and in BALB/3T3 cell transformation: i) The previously reported residual tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) in TNF-/- mice was confirmed by experiments with TNF+/+ and TNF-/- 129/Svj mice of the same strain, using two-stage carcinogenesis experiments. TPA produced tumors in 100% of TNF+/+ and 78% of TNF-/- mice at 20 weeks, and the average number of tumors per mouse was 11.1 in the former group and 2.1 in the latter. Judging from the expression of various inflammatory cytokine genes in TNF+/+ and TNF-/- mice, the residual tumor promoting activity of TPA in TNF-/- mice may be dependent on expression of IL-1alpha and IL-1beta genes. ii) Tumor promotion by TPA and okadaic acid in IL-6+/+ and IL-6-/- C57/BL6 mice was studied, with TPA producing tumors in 57.1% of IL-6+/+ and 40.0% of IL-6-/- mice at 20 weeks, and okadaic acid in 40.0% of IL-6+/+ and 53.3% of IL-6-/- mice. Thus, there was no significant difference between TPA or okadaic acid tumor promotion in either group. In addition, expression of IL-6 gene in skin of both types of mice suggested that IL-6 is not the essential cytokine in tumor promotion, since it can be replaced by other cytokines. iii) In transformed clones of BALB/3T3 cells induced by TNF-alpha alone, IL-1alpha gene expression was induced after transformation by TNF-alpha had occurred, which did not occur in parental cells. Expression patterns of TNF-alpha, IL-1beta, IL-6 and IL-10, along with TGF-beta, were similar in both parental and transformed cells. Considering all these results, we conclude that various cytokines have discrete roles in tumor promotion and cell transformation.     

Gap junctional intercellular communication of primary and asbestos-associated malignant human mesothelial cells

We examined gap junctional intercellular communication (GJIC)of primary human mesothelial cells and cell lines of asbestos-associatedhuman pleural mesotheliomas, and the effect of asbestos andother mineral fibres on these cells. In homologous cultures,the GJIC capacity of six out of seven tumour cell lines wasmarkedly less than for primary mesothelial cells. This defectin GJIC appeared not to be at the expression level of mRNA andprotein of the gene encoding the 43 kDa gap junction protein.In heterologous cocultures of tumour cells and primary mesothelialcells, however, 80–90% of the tumour cell/normal cellcontacts were functional. Exposure of primary mesothelial cellsto TPA, a phorbol ester tumour promoter, resulted in markedinhibition of GJIC, being an action common to numerous tumourpromoters. Such an effect though was not observed with the carcinogenicmesothelioma-inducing mineral fibres chrysotile and amosite,neither with glass wool. These results suggest that a permanentdefect in GJIC capacity is a common feature of human mesotheliomacells, but how mineral fibres are involved in the process ofmesotheliomagenesis is still unclear.