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Human peripheral blood monocytes were cultured and exposed to plant diterpenes, indole alkaloids and polyacetates with various degrees of tumor-promoting activity. The effect of the above-mentioned encounter on monocyte function was examined, as expressed by H2O2 production and lysis of dog erythrocytes by the cells, and the inhibition of 3H-PDBu binding to the monocytes by the various test agents. The most effective reagents in both activation of monocyte function and inhibition of 3H-PDBu binding were 12-0-tetradecanoyl-phorbol 13-acetate (TPA), phorbol 12, 13 dibutyrate, teleocidin, and aplysiatoxin which are known to be strong tumor promoters. Strong stimulation of monocyte function was also exerted by the weak tumor promoters, phorbol 12-retinoate 13-acetate, mezerein and debromoaplysiatoxin. Non-tumor-promoting phorbol diterpenes such as phorbol 12, 13-diacetate, phorbol 12-myristate, phorbol 13-acetate and 4-alpha TPA were 1,000 times less effective than TPA in monocyte stimulation and inhibition of 3H-PDBu binding. These results indicate that stimulation of human monocyte H2O2 production and related cytotoxicity might discriminate effectively between tumor-promoting and non-tumor-promoting reagents.
The induction of mouse skin papillomas in two stages by initiation-promotion protocols has been studied extensively. On the basis of kinetics of induction and morphological observations of apparent progression to carcinomas, it has been argued that the papillomas sequentially advance to carcinomas through promoter-dependent and promoter-independent premalignant stages. Although this hypothesis has been widely accepted, the step-wise development of the same cell population from papilloma to carcinoma has not been established at the cellular level. We show here that some carcinomas which morphologically appear to result from progression of promoter-dependent papillomas do not develop from sequential progression of the same cell population as was found in the original papillomas. Other initiated cells also participate during the growth of these tumors and may replace the original papilloma cells. We also present evidence that some tumors develop directly as promoter-independent papillomas without going through an apparent promoter-dependent papilloma stage.
The kinetics of the irritant response on the mouse ear [erythema measured quantitatively by the irritant dose 50 (ID50)] and its relation to the initiation-promoting activity on the back skin of NMRI mice of some polyfunctional diterpene esters were investigated: (1) A very rapid and transient erythema response is associated with low or absent promoting activity (2) an early and more persistent erythema response in most cases is associated with moderate-to-high promoting activity, and (3) a relatively late onset but persistence up to at least 24 h of the erythema response is associated with high promoting activity. These results may indicate a relationship between the kinetics of the erythema response and the initiation-promoting activity of diterpene esters. The comparatively low promoting activity of esters carrying polyunsaturated acyl functions may indicate the importance of pharmacological parameters such as bioavailability and stability in the biological system, drug-receptor interaction, and "intrinsic activity" of the diterpene esters.
Tryptophan (trp) mRNA synthesis from the authentic trp promoter (Ptrp) is apparently arrested upon translation blockage, while trp mRNA synthesis as a result of read-through from the Pl promoter of the N gene in trp phage is not so affected. When translation is blocked at a nonpermissive temperature in the temperature-sensitive mutants of Escherichia coli rel carrying altered ribosomal elongation factors G (strain CP78G) and Ts (strain HAK88), CP78G and HAK88 show relaxed and stringent phenotypes respectively in control of RNA synthesis. Under conditions causing translation blockage in both mutants, Pl-promoted synthesis of trp mRNA is not depressed while Ptrp-promoted synthesis of trp mRNA is blocked. The insensitivity of Pl-promoted transcription of the translocated trp operon to stringent control is also confirmed by using a strain 10b6s rel carrying a temperature-sensitive valyl-tRNA synthetase. In contrast to the above observation, transcription of the trp operon from either the Pl and Ptrp promoters in a 10b6r rel infected with trp is inhibited greatly at the nonpermissive temperature. This effect occurs even if the level of trp transcription observed at 30 degrees is already low, thus suggesting that translational machinery is intrinsically abnormal in the rel strain.
Susceptibility of hairless inbred S/RV Cri-ba or Bare mice to skin tumor development with suboptimal doses of 7,12-dimethylbenz[a]anthracene (DMBA), DMBA-TPA two stage protocol and two stage promotion using 12-O-tetradecanoylphorbol-13-acetate (TPA) as sub-stage 1 promoter and mezerein (MEZ) or phorbol retinoate acetate (PRA) as substage 2 promoter was determined. A single application of 40 or 20 nmol DMBA induced 4-5 papillomas per mouse 40 weeks after initiation while no tumors appeared after similar treatment with 10 or 4 nmol DMBA. Dose response studies for DMBA initiation revealed that 10 nmol DMBA dose saturated the sites for initiation in the resting epidermis. In two stage promotion experiments, MEZ was found to be a potent stage 2 promoter, while PRA acted as a weak complete promoter.
Derivatives of the Escherichia coli drug-resistance plasmid pBR316 have been constructed which act as molecular probes for promoter-containing DNA restriction fragments from various prokaryotic genomes. The plasmids, designated pBRH1 and pBRH3B, contain a unique EcoRI restriction site located within the promoter for the tetracycline resistance (Tcr) gene. This site was created by the insertion of a chemically synthesized octanucleotide, containing the EcoRI cleavage sequence, into the HindIII site of pBR316. Base-pair alterations within the Tc promoter produced by this insertion resulted in a substantial reduction (pBRH3B) or elimination (pBRH1) in ability of these plasmids to confer Tc resistance to the host strain. Cloning of EcoRI-cleaved foreign DNA fragments into the EcoRI site of these plasmids allows for the isolation of recombinant transformants with Tcr levels greater than that of the plasmid vector. Further characterization of these recombinant plasmids demonstrates that the Tcr phenotype is dependent upon the orientation of the inserted fragment, but not on the molecular weight. We have concluded that these fragments carry promoters which, in the proper orientation, allow for the transcription of the Tcr gene. The utility of these "promoter-probe" plasmids lies in the ability to select for promoter-containing DNA fragments by insertional activation of the Tcr gene.
Previous studies in our laboratory have shown that chemical treatments may induce increases in proliferin gene family mRNA accumulation in cultured murine embryonic cells. Proliferin inductions are highly correlated with subsequent promotional outcomes during two-stage focus-formation assays in C3H/10T1/2 cell cultures. In work reported here, the strong affiliation between these two responses was further validated after treating cells with di-n-butyltin dichloride which is a polyvinyl chloride (PVC) plastic additive that often contaminates food and water. Increased proliferin expression and promotion of morphological transformation occurred at similar concentrations. Promotion of transformation was detected at di-n-butyltin dichloride concentrations of 80 nM (24 ng/ml) and above, if added to initiated cultures before confluent monolayers had formed. Proliferin induction and morphological transformation were both reduced in confluent cultures treated with di-n-butyltin dichloride, as compared to subconfluent cultures. Proliferin expression measured in near-confluent cultures was induced up to 10-fold during the 36-hr period following di-n-butyltin dichloride exposure and was accompanied by increased accumulation of transcripts from many genes regulated by oxidative stresses, growth-inducing agents, and/or other promoting agents (asbestos, superoxide radicals ). Di-n-butyltin dichloride-induced mRNA species included members of the fos and jun proto-oncogene families, c-myc, egr1, ribonucleotide reductase (R2 subunit), odc, macrophage chemotactic protein/je, hsp70, metallothionine IIA, c-sod and mn-sod. The observed patterns of RNA accumulation suggested that a small subset of mRNA species, including proliferin, exhibit regulatory behaviour as a response to dissimilar agents or conditions that promote focus-formation in C3H/10T1/2 cultures. Plausible predictions of promotional effects in two-stage morphological transformation assays can be made from gene-expression responses to test agents.
The effects of the organochlorine (OC) liver tumor promoter heptachlor epoxide (HE; 0, 0.1, 1, 10, and 50 microM) on several cellular tumor promoter-sensitive parameters were studied in mouse 1c1c7 hepatoma cells in an effort to identify the most sensitive biomarker for OC promoter exposure and the critical pathway and target of OC promoters. The levels of Ca2+ in the endoplasmic reticulum (ER) store, connexin43 (Cx43), PLCgamma(1), nPKCvarepsilon, and AP-1 DNA binding in nucleus were studied to screen for effects induced by submicromolar HE levels. While all the parameters tested elicited effects, particulate PLCgamma(1) and AP-1 DNA binding were found to be the most sensitive parameters affected by HE on both dose and temporal bases. Their levels were increased with 10- to 100-fold lower HE concentrations than were required to affect nPKCvarepsilon or Cx43. Further, with the lower HE dosages, particulate PLCgamma(1) and nuclear AP-1 were positively modulated by HE after 1 h versus 3 or 72 h for nPKCvarepsilon and Cx43. Ca2+ store depletion was probably the third most sensitive parameter, after AP-1 and PLCgamma(1). These results suggest the tyrosine kinase growth factor receptor pathway is the probable critical pathway for HE-induce tumor promotion with the critical target most likely being upstream of PLCgamma(1) and AP-1. This work also demonstates that upon exposure to a tumor promoter such as HE, many hepatocellular effects or changes result, suggesting that a cellular-program shift occurs similar to that described by the resistant hepatocyte model after exposure to a carcinogen or enzyme inducer.
The induction of edema in the mouse ear has been established as a reliable in vivo assay for tumor promoting compounds and for inhibitors of tumor promotion. Apparently all skin tumor promoting compounds induce an edema, and all inhibitors of a tumor promoter-induced edema are most likely inhibitors of skin tumor promotion. No exception to this rule has been found as yet. Besides the application of this assay in the screening of compounds, the assay allows comparison of various compounds (e.g. complete and incomplete tumor promoters) with respect to their kinetics and dosage response in the induction of edema.
Promotion is the exacerbation by certain esterase inhibitors (organophosphates, organophosphinates, sulfonyl halides, carbamates and thiocarbamates) of the clinical and morphological expression of toxic and traumatic axonopathies. Promotion is believed to interfere with mechanisms of compensation/repair of the nerves. The target of promotion is unknown but there are indications that it might be similar and/or linked to neuropathy target esterase (NTE), which is the molecular target of organophosphate-induced delayed polyneuropathy (OPIDP). OPIDP is the model axonopathy used to characterize promotion. NTE is defined as the activity resistant to paraoxon (40 microM) and sensitive to mipafox (50 microM). An esterase activity sensitive to higher concentrations (1 mM) of mipafox was identified in the nervous system homogenate, and its inhibition correlated with promotion. An activity with similar characteristics was present in the soluble fraction of peripheral nerves and could be physically separated (about 60 kDa). Identification and characterization of the target of promotion might be helpful in understanding the mechanism(s) of compensation and repair of the peripheral nervous system.
Dietary casein cooked at 180 degrees C promotes the growth of aberrant crypt foci and colon cancer in rats initiated with azoxymethane. We speculated that promotion was due to a product that could be extracted by a solvent, such as 5-hydroxymethyl-2-furaldehyde (HMF), with tumor- promoting activity or the carcinogenic heterocyclic aromatic amines (HAA). This hypothesis was tested by extracting cooked casein with solvents and water. The extracts were then 1) assayed by high-performance liquid chromatography for HMF and HAA, 2) measured for mutagenicity on a frame-shift-sensitive strain of Salmonella typhimurium, and 3) fed for 100 days to azoxymethane-initiated rats to test the promoting effect on aberrant crypt foci. Data show that 1) no HMF or HAA was detected in cooked casein, 2) no mutagenicity was detected on strain TA98, with or without metabolic activation, and 3) promotion was not associated with the extracts but with the cooked casein residue. Therefore the promotion by cooked casein would not appear to be associated with a product that can be extracted by solvents.
The ring hydrolysis products of the multi-tissue tumor promoting barbiturates, phenobarbital (PB) and barbital (BB), were fed to F344/NCr male rats previously given a single initiating injection of N-nitrosodiethylamine. Ethylphenylacetylurea (EPAU) and diethylacetylurea (EEAU), derived, respectively, from PB and BB, both promoted development of hepatocellular adenomas, but were much weaker in this respect than PB. Both acetylureas were also selective inducers of P-450IIB1-mediated benzyloxyresorufin O-dealkylase activity, but both were much less potent inducers than PB. Neither EPAU nor EEAU had an effect on tumor development in the thyroid, unlike both PB and, as shown previously, BB. EEAU, but not EPAU, strongly promoted development of renal cortical epithelial tumors. The opening of the barbiturate heterocyclic ring and the subsequent decarboxylation to yield the dialkylacetylurea analogs thus resulted in compounds displaying a marked reduction in liver tumor promoting activity. EEAU appears to possess an ability to promote renal neoplasms similar to that of its parent compound, BB. Ring opening appears to be accompanied by loss of promoting activity for thyroid follicular epithelium.
Sapintoxin A (SAP A) and 12-deoxyphorbol 13-phenylacetate (DOPP), are two biologically active but non-tumour-promoting phorbol esters that potently bind to and activate the phorbol ester receptor, protein kinase C (PKC). SAP A and DOPP cause a dose-dependent increase in the phosphorylation of an 80 kd (80K) substrate protein for PKC in Swiss 3T3 cells. A similar dose-response effect was seen with sapintoxin D (SAP D), the stage 2 promoting analogue of 12-O-tetradecanoylphorbol-13-acetate and the complete promoter phorbol 12,13-dibutyrate (PDB). The doses resulting in a half maximal phosphorylation of this protein (Ka) were 20 nM (SAP A), 45 nM (DOPP), 23 nM (SAP D) and 37 nM (PDB). Both non-promoting and promoting phorbol esters induced a dose-dependent inhibition of [125I]epidermal growth factor (EGF) binding to its receptor in Swiss 3T3 cells. The doses required for 50% inhibition of binding (Ki) were: 8 nM (SAP A), 16 nM (DOPP), 14 nM (SAP D) and 17 nM (PDB). The results clearly demonstrate that induction of phosphorylation of the 80K phosphoprotein and inhibition of [125I]EGF binding in Swiss 3T3 cells following exposure to phorbol esters is independent of the tumour-promoting activity of these compounds. The fact that SAP A, DOPP, SAP D and PDB are mitogenic for a variety of cell types and that exposure to these compounds leads to 80K phosphorylation and inhibition of [125I]EGF binding, suggests that these early biological events may play a role in the mitogenic response induced by these compounds.
Previous work from our laboratory demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) or a synthetic diacylglycerol induced significantly higher epidermal ornithine decarboxylase (ODC) activity in C57BL/6 than in DBA/2 mice. To understand further the genetic basis for this strain difference, two tumor promoters were evaluated for their effects on epidermal ODC activity: teleocidin, which activates protein kinase C (PKC); and 1,8-dihydroxyl-3-methyl-9-anthrone (chrysarobin), which does not. In addition, the ODC induction response in B6D2F1 offspring and BXD recombinant inbred (RI) strains was examined following multiple treatments with TPA. A single topical application of teleocidin to mouse dorsal skin led to the hyperinduction of epidermal ODC activity in C57BL/6 mice. In contrast, while chrysarobin induced epidermal ODC activity, no significant differences in the magnitude of this response were observed in SENCAR, DBA/2 or C57BL/6 mice. Consistent with our previous findings, the magnitude of ODC induction by teleocidin in these three mouse lines (C57BL/6 greater than SENCAR greater than DBA/2) did not correlate with their susceptibility to tumor promotion by TPA (SENCAR greater than DBA/2 greater than C57BL/6). ODC activity induced by multiple application of TPA in B6DF1 mice, whose susceptibility to phorbol ester tumor promotion is inherited as an incomplete dominant trait, was comparable to that induced in C57BL/6 mice at all the doses examined. Cluster analysis of TPA-induced ODC activity in BXD RI strains allowed us tentatively to group them into four or five phenotypes and to estimate a minimum of two genetic loci controlling TPA-induced ODC activity. Furthermore, in BXD RI strains, there was no apparent relationship between the magnitude of ODC induction and responsiveness to tumor promotion or sustained hyperplasia. Collectively, these results suggest that hyperinducibility of ODC in response to PKC-activating tumor promoters is inherited as an autosomal dominant trait, and that genetic determinants for ODC induction, at least in C57BL/6 and DBA/2 mice, appear completely independent of those controlling tumor promotion susceptibility.
Reactive oxygen species (ROS) have been implicated as being involved in tumor promotion processes. However, the mechanism by which ROS modulate tumor promotion has not as yet been elucidated. In this report, we show that phorbol ester-type tumor promoters (12-O-tetradecanoylphorbol-13-acetate [TPA], mezerein and 12-O-retinoylphorbol-13-acetate [RPA]), which vary in their in vivo potencies, also differ in their effect on formation of hydrogen peroxide (H2O2) and oxidation of normal bases to 5-hydroxymethyl-2'-deoxyuridine [HMdU] and 8-hydroxyl-2'-deoxyguanosine [8-OHdG] in the DNA of SENCAR mouse epidermis, though they are equipotent in causing infiltration of polymorphonuclear leukocytes (PMNs). Treatment of SENCAR mice with the chemopreventive agents (-)-epigallocatechin gallate or tamoxifen (6.5 nmol) prior to application of TPA (6.5 nmol) diminished PMN infiltration, and formation of H2O2, HMdU and 8-OHdG. These results strengthen the evidence that ROS are involved in tumor promotion, and that generation of ROS and the subsequent oxidative DNA modification are related to the tumor-promoting potencies of the different phorbol ester-type promoters.
The role of reactive oxygen (RO) in the promotion of neoplastic transformation of JB6 mouse epidermal cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated using inhibitors of RO itself or RO generating systems of seven different types. Bovine erythrocyte CuZn superoxide dismutase (SOD) maximally decreased anchorage-independent (AI) colony induction by TPA in semi-solid agar in a dose-dependent manner to 10% of TPA control level. The inhibitory effect was specifically on induction of transformation, not expression of transformation. Copper (II) (3,5-diisopropylsalicylic acid)2, which exhibits biomimetic SOD activity, was also effective. Two enzyme eliminators of H2O2, catalase and glutathione peroxidase, failed to prevent TPA-promotion. Among three hydroxyl radical scavengers, D-mannitol and Na-benzoate were moderately active but tetramethylurea did not specifically inhibit AI colony induction by TPA. A quencher of singlet oxygen, 1,4-diazobicyclo-[2,2,2]octane was also inactive. Antioxidants blocked AI transformation by TPA moderately (n-propyl gallate and tannic acid) or weakly (BHA). BHT did not specifically inhibit promotion of transformation. The effects of three inhibitors of the arachidonic acid cascade were examined. NDGA and quercetin (lipoxygenase inhibitors) were moderately active but indomethacin (cyclooxygenase inhibitor) was much less active. Based on these results, we suggest that superoxide anion (O2-.) is required for promotion of transformation by TPA. H2O2 and 1O2 appear not to be required. Hydroxyl radicals and lipid peroxides, possibly associated with O2-. action or formed in the course of oxidative metabolism of arachidonic acid also appear to be required but to a lesser extent. Products of the lipoxygenase pathway of arachidonic acid metabolism but not the cycloxygenase pathway may be important in promotion of transformation by TPA in JB6 mouse epidermal cells. The epidermal cells themselves can be both the source of and the target of the reactive oxygen in promotion.