Tumor Promoter 12-O-tetradecanoylphorbol-13-acetate Research Articles

Anti-tumor promoting activity of Dryopteris phlorophenone derivatives.

As a part of screening studies for cancer chemopreventive agents (anti-tumor promoters) 33 Dryopteris phlorophenone derivatives have been evaluated. The compounds tested comprised of monomeric acylphloroglucinols (e.g. desaspidinol, aspidinol) as well as dimeric (e.g. aspidin, desaspidin), trimeric (e.g. filixic acids), and tetrameric (e.g. dryocrassin) phlorophenone, wherein hexacyclic rings are bound together by a methylene bridge. These compounds were examined for their in vitro anti-tumor promoting effect on Epstein-Barr virus antigen activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The two dimeric compounds aspidin and desaspidin, which were found to be the most active among the tested phlorophenones, were also examined in vivo on two stage mouse skin carcinogenesis, and found to show significant inhibitory effect on 7,12-dimethylbenz[alpha]anthracene (DMBA)-TPA tumor promotion.

Protective effect of all trans retinoic acid against tumor promotion and progression in low- and high-risk protocols of mouse skin chemical carcinogenesis

In this study, we assessed the protective effects of all trans retinoic acid (RA) against skin tumor promotion and progression in three different protocols of mouse skin multistage carcinogenesis. Under these protocols, where papillomas on the skin of SENCAR mice were induced for their low- and high-probability of conversion to malignant carcinomas, pre-application of RA (10 mu g/mouse/application) to that of tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or mezerein (MEZ) in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated mouse skin resulted in a highly significant protection against skin tumor promotion. In terms of tumor incidence, multiplicity and volume/mouse, at the termination of the experiment at 20 weeks, RA showed 35-80%, 67-83% and 70-98% protection, respectively. While the effect of RA was profound in all the protocols, maximum affectivity was evident in high-risk papilloma induction protocol where DMBA was used as tumor initiator and one week later TPA was applied once a week for five weeks. In tumor progression studies, at 20 weeks of standard low- and high-risk protocols, when papilloma yield is stabilized, animals which did not receive RA in different protocols were divided into two groups and topically treated twice weekly either with acetone or RA (10 mu g). These treatments were continued for an additional 29 weeks. During these treatments, all suspected carcinoma formation was recorded and carcinomas were verified histologically at the termination of the experiment. In each case, RA showed remarkable protective effects against percentage of mice with carcinomas (35-45% protection), number of carcinomas per mouse (50-63% protection) and the rate of malignant conversion (50-63% protection). The results of the present study clearly demonstrate that irrespective of the risk for skin carcinogenesis, RA is capable of affording protection against the induction of nonmalignant lesions and their subsequent conversion to malignancy.

Camptothecin post-treatments inhibit the biochemical events linked to the tumor-promoting component of carcinogenesis in mouse epidermisin vivo

20(S)-Camptothecin (CPT), a topoisomerase I inhibitor specifically toxic toward S-phase cells, was tested topically for its ability to inhibit the biochemical markers of skin tumor promotion. CPT has no or very little inhibitory effect on the covalent binding of an initiating dose of 7,12-dimethylbenz-[a]anthracene (DMBA) to DNA at 24 hr, but CPT post-treatments remarkably inhibit stimulations of DNA synthesis caused by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 16 hr and a carcinogenic dose of DMBA at 7 days. CPT is a much more potent inhibitor if it is applied 10-14 hr after TPA or 4-6 days after DMBA, when DNA synthesis starts being stimulated after the periods of early inhibition caused by TPA and DMBA. When applied 12 hr after the tumor promoter, the ability of 3-3,000 nmol of CPT to inhibit TPA-stimulated DNA synthesis at 16 hr is dose-dependent. A single dose of 500 nmol of CPT inhibits the entire time course for the stimulation of DNA synthesis observed 16-64 hr after TPA. CPT also reduces the various DNA responses to chronic TPA treatments and structurally different non-TPA-type tumor promoters. CPT may indirectly decrease the ornithine decarboxylase-inducing activity of multiple TPA treatments because it can inhibit the stimulation of RNA synthesis by this compound. However, CPT fails to alter TPA-stimulated hydroperoxide production in relation to its inability to inhibit TPA-stimulated protein synthesis. On an equal dose basis, topotecan and 10-hydroxycamptothecin are more and less effective than CPT, respectively, whereas 10,11-methylenedioxycamptothecin is much more potent than its parent compound at inhibiting the DNA response to TPA. A single dose of 400 nmol of CPT has no effect on tumor initiation when applied 4 hr before or 1 hr after a single subcarcinogenic dose of DMBA. In contrast, 400 nmol of CPT chronically applied 1 hr before or 24 hr after each treatment with TPA remarkably inhibits the complete tumor-promoting activity of this agent. CPT post-treatments also inhibit the respective activities of TPA and mezerein in the 1st and 2nd stages of skin tumor promotion.

Determination of gap junctional intercellular communication by capacitance measurements

Electrical coupling between cells is usually measured using the double patch-clamp technique with cell pairs. Here, a single patch-clamp technique that is not limited to cell pairs is described to determine electrical coupling between cells. Capacitance measurements in clusters of normal rat kidney (NRK) fibroblasts were used to study intercellular communication. In the whole-cell patch-clamp configuration capacitive transients were evoked by applying small voltage pulses. Total membrane capacitance was calculated from these capacitive transients after determination of access resistance, membrane conductance, and the decay constant of the transients, or alternatively by integrating the current transient. We found that in clusters of one to ten cells, membrane capacitance increased linearly with cell number, showing that the cells are electrically coupled. Membrane conductance of the cluster of cells also increased, as expected for cells that are well coupled. In subconfluent and confluent cultures, high membrane conductances together with large capacitive transients were observed, indicative of electrical coupling. Capacitance could only be determined qualitatively under these conditions, due to space clamp problems. In the presence of the gap junctional inhibitors halothane, heptanol or octanol, capacitance of all clusters of cells fell to single-cell levels, showing a complete uncoupling of the cells. The tumour promoter 12-O-tetradecanoylphor-bol-13-acetate (TPA) also uncoupled the cells completely, within 10 min. We conclude that capacitance measurements can provide a useful tool to study changes in intercellular communication in clusters of cells.

Protein kinase C isotypes required for phorbol-ester induction of stromelysin-1 in rat fibroblasts

The phorbol-ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of the metalloproteinase stromelysin in fibroblasts in vivo and in several cultured cell lines. Rat-1 and Rat-2 fibroblasts, however, do not respond to TPA stimulation by induction of stromelysin gene activity, although collagenase promoter-mediated activity is induced threefold by TPA treatment in these cells. We determined that rat fibroblasts expressed protein kinase C(PKC)alpha, PKCdelta, PKCepsilon, and PKCzeta but neither the mRNA nor the protein for PKCbeta. When Rat-2 fibroblasts were stably transfected with an expression vector producing PKCbeta, however, TPA treatment of these variants resulted in a 3.1-fold induction of stromelysin promoter-mediated luciferase activity compared with a 1.3-fold induction in parental Rat-2 cells (P<0.002). Transient transfection of PKCepsilon produced a small but significant increase in TPA-stimulation of both stromelysin- and collagenase-mediated gene expression. These results suggest that there are PKC isotype-specific signaling pathways that can differentially regulate matrix metalloproteinase gene expression.

Determination of gap junctional intercellular communication by capacitance measurements

Electrical coupling between cells is usually measured using the double-patch-clamp technique with cell pairs. Here, a single patch-clamp technique that is not limited to cell pairs is described to determine electrical coupling between cells. Capacitance measurements in clusters of normal rat kidney (NRK) fibroblasts were used to study intercellular communication. In the whole-cell patch-clamp configuration capacitive transients were evoked by applying small voltage pulses. Total membrane capacitance was calculated from these capacitive transients after determination of access resistance, membrane conductance, and the decay constant of the transients, or alternatively by integrating the current transient. We found that in clusters of one to ten cells, membrane capacitance increased linearly with cell number, showing that the cells are electrically coupled. Membrane conductance of the cluster of cells also increased, as expected for cells that are well coupled. In subconfluent and confluent cultures, high membrane conductances together with large capacitive transients were observed, indicative of electrical coupling. Capacitance could only be determined qualitatively under these conditions, due to space clamp problems. In the presence of the gap junctional inhibitors halothane, heptanol or octanol, capacitance of all clusters of cells fell to single-cell levels, showing a complete uncoupling of the cells. The tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also uncoupled the cells completely, with 10 min. We conclude that capacitance measurements can provide a useful tool to study changes in intercellular communication in clusters of cells.

Enhancing effect of ultraviolet A on ornithine decarboxylase induction and dermatitis evoked by 12-o-tetradecanoylphorbol-13-acetate and its inhibition by curcumin in mouse skin.

Previous studies have demonstrated appreciable tumor induction in mouse skin by daily irradiation with high-power long-wavelength ultraviolet A (UVA). The aim of the present study was to examine the enhancing effects of UVA on changes in mouse skin mediated by the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA) by measurement of ornithine decarboxylase (ODC) activity and morphometric analysis. In addition, we examined the inhibitory effects of curcumin, a component of turmeric, on these changes. ODC activity in the epidermis of CD-1 mice was determined by the method of Russell and Snyder. Epidermal and dermal thickness, and the number of dermal infiltrating inflammatory cells were quantified using a computer-assisted image analyzer. A combination of topical TPA application and UVA irradiation produced a greater increment of ODC activity at 4 h than TPA alone (p < 0.05). Histopathologically, TPA plus UVA tended to increase the dermal infiltrating inflammatory cells in contrast to TPA alone. Pretreatment of mice with curcumin significantly abrogated the TPA-induced changes in ODC activity and the dermal infiltrating inflammatory cells as well as the TPA plus UVA-mediated enhancement of these changes. Our data indicate that UVA irradiation (18.72 J/cm2) significantly enhances ODC induction at an early stage (4-6 h) after topical application of TPA, and aggravates the dermatitis elicited by TPA. Pretreatment with curcumin significantly inhibits these enhancing effects.

Dietary retinoic acid inhibits mouse skin carcinogenesis irrespective of age at initiation.

In the two-stage protocol of skin carcinogenesis, the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) is applied to the skin of mice at around seven weeks of age. We previously performed DMBA initiation at three weeks of age to study the effect of pharmacological (30 micrograms/g diet) dietary retinoic acid (RA) on skin carcinogenesis. In this study we asked whether dietary pharmacological RA is equally effective against skin carcinogenesis when mice are initiated with (DMBA) at 7 weeks of age and then subjected to weekly applications of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or mezerein (MEZ) for 20 weeks. Similar to the three-week initiation protocol, high dietary RA inhibited papilloma incidence and yield in MEZ- but not in TPA-promoted female SENCAR mice. In addition, carcinoma incidence and yield were decreased by high dietary RA in TPA- as well as MEZ-treated mice. These data demonstrate that the high dietary RA diet is as effective in inhibiting papilloma and carcinoma formation when the DMBA is applied at seven weeks of age as at three weeks.

Anti-tumor-promoting activities of triterpenoids from ferns. I.

To search for possible anti-tumor-promoters (cancer chemopreventive agents), we carried out primary screening of 23 triterpenoid hydrocarbons (1-23) isolated from ferns using an in vitro synergistic assay system. Of these triterpenoids, hop-17(21)-ene (2), neohop-13(18)-ene (3), neohop-12-ene (4), taraxerane (17), multiflor-9(11)-ene (18), multiflor-8-ene (19), glutin-5(10)-ene (21) and taraxastane (23) exhibited remarkable inhibitory effects on Epstein-Barr virus (EBV) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Further, compounds 2 and 3 exhibited remarkable anti-tumorpromoting effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test using 7,12-dimethybenz[a]anthracene (DMBA) as an initiator and TPA as a promoter.

All-trans beta-carotene enhances mitogenic responses and ornithine decarboxylase activity of BALB/c 3T3 fibroblast cells induced by tumor promoter and fetal bovine serum but suppresses mutagen-dependent umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002).

Although previous epidemiological studies have indicated that beta-carotene is an important agent for the chemical prevention against carcinogenesis, a recent prospective study has strikingly suggested that supplementation with beta-carotene significantly increased the incidence of some types of cancer (The alpha-Tocopherol and beta-Carotene Cancer Prevention Study Group, New Engl. J. Med., 330 (1994) 1031-1035). To analyze the discrepancy of this problem, the authors analyze the effects of beta-carotene on biochemical and biological events associated with carcinogenesis by in vitro experiments. (1) All-trans beta-carotene enhanced the proliferation and DNA synthesis of BALB/c 3T3 cells induced by a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and fetal bovine serum, although beta-carotene itself did not show mitogenic activity. (2) All-trans beta-carotene caused a remarkable stimulation for the early induction of ornithine decarboxylase (ODC) activity after the stimulation of TPA and fetal bovine serum. (3) All-trans beta-carotene exhibited significant antimutagenic activity which suppresses umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) induced by a typical mutagen, 2-aminoanthracene (2-AA). These experimental results suggest that all-trans beta-carotene might cause beneficial and harmful effects on different phases of carcinogenesis.

Estrogen and phorbol esters regulate amphiregulin expression by two separate mechanisms in human breast cancer cell lines

The actions of 17 beta-estradiol (E2) and protein kinase C (PKC) appear to converge in the regulation of expression of certain growth modulatory genes, such as the growth factor amphiregulin (AR). AR is known to modulate cell growth by binding to the epidermal growth factor receptor. In the current report we established the mechanisms of the PKC-activating phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the steroid hormone E2 on the induction of AR expression in human breast carcinoma cell lines. TPA (100 nM) and E2 (1 nM) induce AR messenger RNA (mRNA) expression by 6- to 8-fold and 3- to 6-fold, respectively, in a time- and dose-dependent manner. In addition, immunoreactive AR protein is induced by both TPA and E2 by 6- to 8-fold and 2- to 4-fold, respectively. The PKC-modulating drugs, bryostatin and H-7, and antiestrogens (ICI 164,384 and 4-hydroxytamoxifen) interfere with AR induction by TPA and estrogen, respectively. The effects of TPA and E2 on the induction of AR mRNA were both closely associated with enhanced transcription of the AR gene. However, TPA had an additional effect at the posttranscriptional level by stabilizing the AR mRNA. The protein synthesis inhibitor, cycloheximide, prevented AR induction by TPA, suggesting that a component of the TPA induction of AR is indirect and dependent upon protein synthesis. Conversely, the E2 induction of AR transcription was found to be a direct response, independent of protein synthesis. The results presented herein thus demonstrate that TPA and E2 are able to stimulate AR gene transcription by two separate mechanisms.

Estrogen and phorbol esters regulate amphiregulin expression by two separate mechanisms in human breast cancer cell lines.

The actions of 17 beta-estradiol (E2) and protein kinase C (PKC) appear to converge in the regulation of expression of certain growth modulatory genes, such as the growth factor amphiregulin (AR). AR is known to modulate cell growth by binding to the epidermal growth factor receptor. In the current report we established the mechanisms of the PKC-activating phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the steroid hormone E2 on the induction of AR expression in human breast carcinoma cell lines. TPA (100 nM) and E2 (1 nM) induce AR messenger RNA (mRNA) expression by 6- to 8-fold and 3- to 6-fold, respectively, in a time- and dose-dependent manner. In addition, immunoreactive AR protein is induced by both TPA and E2 by 6- to 8-fold and 2- to 4-fold, respectively. The PKC-modulating drugs, bryostatin and H-7, and antiestrogens (ICI 164,384 and 4-hydroxytamoxifen) interfere with AR induction by TPA and estrogen, respectively. The effects of TPA and E2 on the induction of AR mRNA were both closely associated with enhanced transcription of the AR gene. However, TPA had an additional effect at the posttranscriptional level by stabilizing the AR mRNA. The protein synthesis inhibitor, cycloheximide, prevented AR induction by TPA, suggesting that a component of the TPA induction of AR is indirect and dependent upon protein synthesis. Conversely, the E2 induction of AR transcription was found to be a direct response, independent of protein synthesis. The results presented herein thus demonstrate that TPA and E2 are able to stimulate AR gene transcription by two separate mechanisms.

Inhibition of 12-O-Tetradecanoylphorbol-13-Acetate and Other Skin Tumor-Promoter-Caused Induction of Epidermal Interleukin-1α mRNA and Protein Expression in SENCAR Mice by Green Tea Polyphenols

Recent studies have shown that topical application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to murine skin results in increased expression of the highly inflammatory cytokine interleukin (IL)-1 alpha in the epidermis. This has led to the suggestion that IL-1 alpha directly or indirectly mediates the inflammatory and hyperplastic responses elicited by TPA and possibly by other skin tumor promoters. In the current study, we investigated the effect of skin application of a polyphenolic fraction isolated from green tea (GTP) to SENCAR mice on skin tumor-promoter-caused induction of cutaneous edema and hyperplasia, and IL-1 alpha mRNA expression. Pretreatment of the skin with GTP 30 min before that of anthralin, benzoyl peroxide, mezerein, and TPA resulted in a significant (p < 0.05) inhibition of cutaneous edema and epidermal hyperplasia caused by each of these tumor promoters. Northern blot analysis indicated that topical application of TPA, anthralin, mezerein, or benzoyl peroxide to SENCAR mice resulted in an increased expression of epidermal IL-1 alpha mRNA. Pretreatment of the skin with GTP or individual epicatechin derivatives (ECDs) present therein, 30 min before that of TPA, resulted in a significant inhibition of enhanced expression of epidermal IL-1 alpha mRNA caused by skin application of TPA. These inhibitory effects were found to be dependent on the dose of GTP. Among four epicatechin derivatives present in GTP, (-)-epicatechin-3-gallate and (-)-epigallocatechin-3-gallate were more effective than (-)-epigallocatechin and (-)-epicatechin in affording this inhibition. Preapplication of GTP was also found to afford inhibition against anthralin-, benzoyl peroxide-, and mezerein-caused increased expression of epidermal IL-1 alpha mRNA and protein. Our study suggests that the inhibition of tumor-promoter-induced IL-1 alpha mRNA and protein expression in mouse epidermis by green tea in combination with other inhibitory effects may be responsible for the anti-tumor-promoting and anti-inflammatory effects of GTP.

Differential pathways of insulin action upon the hepatitis B surface antigen gene expression and cell proliferation in human hepatoma cells

We have shown previously that insulin at the physiological concentration suppresses hepatitis B surface antigen (HBsAg) gene expression in cultured human hepatoma Hep3B cells, and this suppression phenomenon is concomitant with the stimulation of cell proliferation. We have now examined whether these two distinct insulin actions on the Hep3B cells are mediated through the same or different signaling pathways. After prolonged treatment with high concentration of tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), the protein kinase C-alpha (PKC-alpha) level in the Hep3B cells was diminished and could not be detected by Western blot analysis. Under this condition, TPA treatment has no effect on the number or affinity of the insulin receptor on Hep3B cells. However, insulin-stimulated cell proliferation was completely abolished in the PKC-alpha depleted cells. In contrast, insulin still suppressed HBsAg gene expression with the same ED50 (approximately 0.5 nM) as the control cell. The induction of proto-oncogene egr-1 (early-growth-regulatory-1) by insulin and TPA under similar conditions were also examined. Both insulin and TPA stimulated egr-1 gene expression up to 10-fold in the control cell, but neither of these two agents showed any effect on egr-1 gene expression in the PKC-alpha down-regulated Hep3B cells. These observations indicate that the PKC-alpha may be involved in the insulin induced egr-1 expression and cell proliferation but not in the insulin suppressed HBsAg gene expression in human hepatoma cells.

Differential pathways of insulin action upon the hepatitis B surface antigen gene expression and cell proliferation in human hepatoma cells.

We have shown previously that insulin at the physiological concentration suppresses hepatitis B surface antigen (HBsAg) gene expression in cultured human hepatoma Hep3B cells, and this suppression phenomenon is concomitant with the stimulation of cell proliferation. We have now examined whether these two distinct insulin actions on the Hep3B cells are mediated through the same or different signaling pathways. After prolonged treatment with high concentration of tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), the protein kinase C-alpha (PKC-alpha) level in the Hep3B cells was diminished and could not be detected by Western blot analysis. Under this condition, TPA treatment has no effect on the number or affinity of the insulin receptor on Hep3B cells. However, insulin-stimulated cell proliferation was completely abolished in the PKC-alpha depleted cells. In contrast, insulin still suppressed HBsAg gene expression with the same ED50 (approximately 0.5 nM) as the control cell. The induction of proto-oncogene egr-1 (early-growth-regulatory-1) by insulin and TPA under similar conditions were also examined. Both insulin and TPA stimulated egr-1 gene expression up to 10-fold in the control cell, but neither of these two agents showed any effect on egr-1 gene expression in the PKC-alpha down-regulated Hep3B cells. These observations indicate that the PKC-alpha may be involved in the insulin induced egr-1 expression and cell proliferation but not in the insulin suppressed HBsAg gene expression in human hepatoma cells.

Expression of gamma‐glutamyl transpeptidase by renal epithelial cells occurs on a cell‐by‐cell basis and is inhibited by the chronic TPA treatment

Upon attaining a confluent density, populations of the renal epithelial cell line, LLC-PK1, express progressively many properties characteristic of the renal proximal tubule cell, including gamma-glutamyl transpeptidase activity. Expression of transpeptidase activity was inhibited reversibly by chronic treatment with the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA treatment inhibited expression of transpeptidase activity regardless of whether added prior to or following appearance of the activity. Increased transpeptidase activity in postconfluent cell populations was due to an increased enzyme Vmax with no change in substrate Km. TPA-treated cell populations exhibited a low Vmax similar to subconfluent populations. Detection of transpeptidase activity at the individual cell level by enzyme histochemistry demonstrated that near-confluent cell populations possessed few transpeptidase activity-positive cells. Progressive expression of transpeptidase activity in the cell population was due to an increasing proportion of cells in the population possessing transpeptidase activity. There was a parallel increase in the proportion of cells expressing transpeptidase protein, detected by immunofluorescence. TPA treatment inhibited appearance of both transpeptidase activity and transpeptidase protein in virtually all cells of the population. These results demonstrate that expression of transpeptidase activity in populations of LLC-PK1 cells occurs on a cell-by-cell basis and reflects expression of transpeptidase protein. Chronic treatment with TPA inhibits reversibly expression of transpeptidase activity and protein, suggesting a role for protein kinase C in regulating expression of this proximal tubule-specific property.

Okadaic acid co‐induces vimentin expression and cell cycle arrest in MPC‐11 mouse plasmacytoma cells

The effect of the tumor promoter okadaic acid on cell cycle progression and on vimentin expression in MPC-11 mouse plasmacytoma cells was compared with that of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Cell cycle progression of asynchronously grown MPC-11 cells was inhibited by both agents, but, in contrast to the G1 phase arrest caused by TPA, okadaic acid gave rise to G2/M phase and S phase arrest. This effect of okadaic acid was delayed significantly compared to the TPA-caused arrest. Furthermore, okadaic acid was able to induce vimentin expression to an extent comparable to the TPA response. However, vimentin expression was markedly delayed in okadaic acid-treated relative to TPA-treated cells. Another protein phosphatase inhibitor, calyculin A, also induced cell cycle changes and vimentin expression at concentrations at or above 1 x 10(-9) M. Based on these observations, we suggest an involvement of protein phosphatase 1 (possibly also phosphatase 2A and/or other phosphatases) in both the G2/M cell cycle block and the induction of vimentin expression in MPC-11 cells by okadaic acid.

Recombinagenic activity of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in human lymphoblastoid cells.

The potent mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its mutagenic and recombinagenic activity at the heterozygous thymidine kinase (tk +/-) locus and the hemizygous hypoxanthine phosphoribosyltransferase (hprt +/0) locus in the TK6 human lymphoblastoid cell line. TPA at concentrations of 0.01-1.0 micrograms/ml induced a low frequency of tk mutants showing the slow growth phenotype in a dose-dependent manner, but few normal growth tk mutants or hprt mutants. Concentrations of 1.0-10 micrograms/ml TPA induced all three types of mutants. The molecular structure of tk mutants arising spontaneously or induced by 1.0 and 10 micrograms/ml TPA was investigated by Southern hybridization with a human tk cDNA probe: 86% of all mutants arising after incubation with 10 micrograms/ml TPA lost the entire active tk allele, resulting in loss of heterozygosity (LOH), while 71% of spontaneously arising mutants showed LOH. Densitometric analysis indicated that the majority of LOH mutants induced by TPA were homozygous at the tk locus (retained two copies of the mutant allele), consistent with the occurrence of interchromosomal homologous recombination. These results support the hypothesis that tumor promoters such as TPA may increase the rate of chromosomal mitotic recombination and hence facilitate the segregation of recessive mutations. TPA may thus induce a type of genetic instability during the process of tumor promotion that involves enhanced recombinagenic activity.

Differential expression of prostaglandin H synthase isozymes during multistage carcinogenesis in mouse epidermis.

An anti-tumor-promoting effect of indomethacin and related nonsteroidal anti-inflammatory drugs (NSAIDs) as well as the ability of the tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) to increase the level of prostaglandins in murine keratinocytes and mouse epidermis in vivo has been repeatedly documented. Here, the expression of prostaglandin H synthase (PGHS) isozymes, which are major targets of NSAIDs, was investigated in different stages of tumor development in mouse skin. Mouse epidermis in vivo constitutively expressed PGHS-1. PGHS-1 steady-state levels remained unchanged upon induction of acute or chronic epidermal hyperplasia by TPA and in papillomas and carcinomas generated by the initiation-promotion procedure, with 7,12-dimethylbenz[a]anthracene as initiator and TPA as promoter. Thus, the elevated prostaglandin level in the acute hyperplastic epidermis was very likely due to PGHS-2 induction. Repeated applications of TPA resulted in stationary hyperplasia and downregulation of PGHS-2 expression and prostaglandin levels, suggesting that the epidermis had adapted to the TPA stimulus. In papillomas and carcinomas, however, constitutive overexpression of PGHS-2 was found, with a large amount of prostaglandin E2 and prostaglandin F2 alpha. Keratinocyte cell lines corresponding to different stages of tumor development also constitutively over-expressed PGHS-2. Considered with inhibitor studies, these data suggest that PGHS-2 has a critical role in skin carcinogenesis. The anti-tumor-promoting effect of the PGHS inhibitor indomethacin is specifically reversed by prostaglandin F2 alpha, indicating that this prostaglandin type has a significant role in tumor development.

Overexpression of protein kinase C beta 1 in the SW480 colon cancer cell line causes growth suppression.

Using a retroviral vector system we have established derivatives of the E8 subclone of the human colon cancer cell line SW480 that stably overproduce a full-length rat cDNA encoding the beta 1 isoform of protein kinase C (PKC beta 1). In contrast to vectrol control cells, when treated with the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), the overexpressing cell lines displayed a striking increase in doubling time, and a decrease in saturation density. Western blot analysis indicated that treatment with TPA was also associated with translocation and partial downregulation of the exogenous PKC beta 1 in the over-expressor cell lines. These results extend previous evidence that PKC beta 1 can inhibit the growth of the HT29 human colon cancer cell line. The HT29 cells have a normal c-k-ras oncogene but the SW480 cells used in the present study have an activating mutation in this oncogene. Thus PKC beta 1 can function as a suppressor in both types of colon cancer cells.