Ethanol specifically decreases peroxisome proliferator activated receptor beta in B12 oligodendrocyte-like cells

Peroxisome proliferator activated receptors (PPARs) are nuclear receptors that control important genes involved in lipid metabolism. Their role in nerve cells is uncertain, although anomalous myelination of the corpus callosum has been described in the PPARbeta-null mouse, and abnormalities of this tissue have been documented in fetal alcohol syndrome in humans. We report here that ethanol treatment of B12 oligodendrocyte-like cells induces a concentration- and time-dependent decrease in the mRNA and protein levels of PPARbeta, with no effect on PPARalpha or PPARgamma. The effect on PPARbeta is seen as an increase in mRNA degradation, as assessed by run-off assays, due to a significant decrease in PPARbeta mRNA half-life, with no observed changes in intracellular localization. Our results suggest a possible link between PPARbeta function and ethanol-induced abnormal myelination in oligodendrocytes.     

Bezafibrate is a dual ligand for PPARalpha and PPARbeta: studies using null mice

Bezafibrate is a known activator of peroxisome proliferator-activated receptors (PPARs) that can activate both PPARalpha and PPARbeta. To determine the role(s) of these receptors in mediating the biological effects of this chemical, the effect of bezafibrate was examined in PPARalpha-null and PPARbeta-null mice. Wild-type, PPARalpha-null, or PPARbeta-null mice were fed either a control diet or one containing 0.5% bezafibrate for 10 days. Bezafibrate feeding caused a significant increase in liver weight in wild-type and PPARbeta-null mice compared to controls, while liver weight was unchanged in bezafibrate-fed PPARalpha-null mice. Gonadal adipose stores were significantly smaller in wild-type and PPARbeta-null mice fed bezafibrate than in controls, and this effect was not found in similarly fed PPARalpha-null mice. Analysis of liver, white adipose tissue, and intestinal mRNAs showed that bezafibrate caused similar changes of mRNAs encoding lipid metabolizing enzymes in wild-type and PPARbeta-null mice compared to controls. Interestingly, in PPARalpha-null mice, bezafibrate also induced several mRNAs previously thought to be solely controlled by PPARalpha, showing that the effects of this drug are not exclusively modulated by this PPAR isoform. Western blot analysis of liver protein was consistent with changes in mRNA expression showing that the alterations in mRNA expression correlate with protein expression in this tissue. Results from these studies demonstrate that the effect of bezafibrate is mediated in large part by PPARalpha, although some changes in gene expression are dependent on PPARbeta. In contrast to other PPARalpha ligands such as WY-14,643, induction of some target genes by bezafibrate can also be modulated in the absence of a functional PPARalpha.     

Peroxisome proliferator-activated receptor-beta/delta inhibits epidermal cell proliferation by down-regulation of kinase activity

Recent work has shown that peroxisome proliferator-activated receptor beta (PPARbeta) attenuates cell proliferation and skin carcinogenesis, and this is due in part to regulation of ubiquitin C expression. In these studies, the role of PPARbeta in modulating ubiquitin-dependent protein kinase Calpha (PKCalpha) levels and phosphorylation signaling pathways was evaluated. Intracellular phosphorylation analysis showed that phosphorylated PKCalpha and other kinases were lower in wild-type mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) as compared with PPARbeta-null mouse skin. No differences in expression levels of other PKC isoforms present in skin were observed. Lower ubiquitination of PKCalpha was found in TPA-treated PPARbeta-null skin as compared with wild-type, and inhibition of ubiquitin-dependent proteasome degradation prevented TPA-induced down-regulation of PKCalpha. The activity of PKCalpha and downstream signaling kinases is enhanced, and expression of cyclooxygenase-2 (COX-2) is significantly greater, in PPARbeta-null mouse skin in response to TPA compared with wild-type mouse skin. Inhibition of PKCalpha or COX-2 reduced cell proliferation in TPA-treated PPARbeta-null keratinocytes in a dose-dependent manner, whereas it only slightly influenced cell proliferation in wild-type keratinocytes. Combined, these studies provide strong evidence that PPARbeta attenuates cell proliferation by modulating PKCalpha/Raf1/MEK/ERK activity that may be due in part to reduced ubiquitin-dependent turnover of PKCalpha.     

Peroxisome proliferator-activated receptor beta/delta regulates very low density lipoprotein production and catabolism in mice on a Western diet

The results of recent studies using selective agonists for peroxisome proliferator-activated receptor beta (PPARbeta) suggest that this receptor may have a role in regulating levels of serum lipids in animal models of obesity and insulin resistance. To further examine this possibility, serum lipid profiles of mice lacking a functional PPARbeta receptor were determined. PPARbeta-null mice maintained on either normal chow or a 10-week high fat (HF) diet, a condition that has been shown to induce insulin resistance and obesity in mice, have elevated levels of serum triglycerides primarily associated with very low density lipoprotein (VLDL) with no difference in either total cholesterol or phospholipids. Consistent with this finding, PPARbeta-null mice on a HF-diet were shown to have an increased rate of hepatic VLDL production as well as lowered lipoprotein lipase activity in serum compared with wild-type controls. The latter parallels an increase in the hepatic expression of the genes encoding angiopoietin-like proteins 3 and 4 in PPARbeta-null mice on a HF diet, both proteins of which have recently been shown to inhibit lipoprotein lipase (LPL) activity in vivo. Consistent with elevated VLDL production, a marked increase in plasma VLDL apoB48, -E, -AI, and -AII, as well as a sharp depletion of the hepatic lipid stores was also found in PPARbeta-null mice. In addition, PPARbeta-null mice on a HF diet were shown to have increased adiposity, despite lower total body weight. Together, these results indicate a clear role for PPARbeta in regulating levels of serum triglycerides in mice on a high fat Western diet by modulating both VLDL production and LPL-mediated catabolism of VLDL-triglycerides and also suggest a potential therapeutic role for PPARbeta in the improvement of serum lipids in the setting of metabolic syndrome.     

Peroxisome proliferator-activated receptor beta (delta)-dependent regulation of ubiquitin C expression contributes to attenuation of skin carcinogenesis

The role of peroxisome proliferator-activated receptor-beta (PPARbeta) in the molecular regulation of skin carcinogenesis was examined. Increased caspase-3 activity associated with apoptosis was found in the skin of wild-type mice after tumor promotion with 12-O-tetradecanoylphorbol-13-acetate, and this effect was diminished in PPARbeta-null mice. The onset of tumor formation, tumor size, and tumor multiplicity induced from a two-stage carcinogen bioassay (7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate) were significantly enhanced in PPARbeta-null mice compared with wild-type mice. To begin to characterize the molecular changes underlying this PPARbeta-dependent phenotype, microarray analysis was performed and a number of differentially regulated gene products were identified including ubiquitin C. Subsequent promoter analysis, reporter gene assays, site-directed mutagenesis, and electrophoretic mobility shift assays provide evidence that PPARbeta regulates ubiquitin C expression, and that ubiquitination of proteins is influenced by PPARbeta. These results strongly suggest that activation of PPARbeta-dependent target genes provides a novel strategy to inhibit tumor promotion and carcinogenesis.     

PPARbeta/delta selectively induces differentiation and inhibits cell proliferation

Peroxisome proliferator-activated receptor (PPAR) beta-null mice exhibit exacerbated epithelial cell proliferation and enhanced sensitivity to skin carcinogenesis, suggesting that ligand activation of PPARbeta will inhibit keratinocyte proliferation. By using of a highly specific ligand (GW0742) and the PPARbeta-null mouse model, activation of PPARbeta was found to selectively induce keratinocyte terminal differentiation and inhibit keratinocyte proliferation. Additionally, GW0742 was found to be anti-inflammatory due to inhibition of myeloperoxidase activity, independent of PPARbeta. These data suggest that ligand activation of PPARbeta could be a novel approach to selectively induce differentiation and inhibit cell proliferation, thus representing a new molecular target for the treatment of skin disorders resulting from altered cell proliferation such as psoriasis and cancer.     

Effect of chronic continuous or intermittent hypoxia and reoxygenation on cerebral capillary density and myelination

Chronic hypoxia, whether continuous (CCH) or intermittent (CIH), occurs in many neonatal pathological conditions, such as bronchopulmonary dysplasia and obstructive sleep apnea. In this study, we explored the effect of CCH and CIH on cerebral capillary density and myelination. We subjected CD-1 mice starting at postnatal day 2 to either CCH 11% oxygen (O(2)), or CIH 11% O(2) (4-min cycles), for periods of 2 and 4 wk followed by reoxygenation for 4 wk. Mice were deeply anesthetized and perfused. Brains were removed to fixative for 24 h, then paraffin-embedded. Coronal brain sections were taken for analysis. Immunocytochemistry for glucose transporter 1 was used to assess angiogenesis, and Luxol fast blue and fluoromyelin stains were used to assess myelination. Capillary density increased after 2-wk exposure to CIH and CCH. By 4 wk, capillary density increased in both CIH and CCH by 25% and 47%, respectively, in cortex and by 29% and 44%, respectively, in hippocampus (P < 0.05). There was a decrease in myelination in the corpus callosum of mice exposed to CIH (75% of control) and CCH (50% of control) (P < 0.05). Reoxygenation reversed the increased capillary density seen in CCH to normoxic values. However, dysmyelination that occurred in CCH-exposed mice did not show any improvement upon reoxygenation. We conclude that neonatal chronic hypoxia 1) induces brain angiogenesis, which is reversible with reoxygenation, and 2) irreversibly reduces the extent of myelination in the corpus callosum. This potential irreversible effect on myelination in early life can, therefore, have long-term and devastating effects.     

The activity of 2'',3''-cyclic nucleotide 3''-phosphohydrolase in the corpus callosum, subcortical white matter, and spinal cord in infants dying from sudden infant death syndrome

Abstract: The activity of 2',3'-cyclic nucleotide 3'-phos-phohydrolase (CNPase) has been determined in corpus callosum, subcortical white matter, and spinal cord of infants whose death was attributed to the sudden infant death syndrome (SIDS), and compared with enzyme activity in other cases in which the cause of death was not associated with respiratory distress. In nearly half the SIDS cases, CNPase activity and oligodendroglial cell numbers were reduced before the onset of myelination, but only in the corpus callosum. In other SIDS cases, enzyme activity and cell numbers were the same as in non-SIDS cases. If the expression of CNPase activity reflects glioblast differentiation to oligodendrocytes with myelinating potential, then this transformation is abnormal in certain SIDS cases, as also evidenced in cases of prolonged neonatal respiratory insufficiency and gives rise to a subsequent deficit of myelin in the corpus callosum.     

Corpus callosum morphology in capuchin monkeys is influenced by sex and handedness

Sex differences have been reported in both overall corpus callosum area and its regional subdivisions in humans. Some have suggested this reflects a unique adaptation in humans, as similar sex differences in corpus callosum morphology have not been reported in any other species of primate examined to date. Furthermore, an association between various measurements of corpus callosum morphology and handedness has been found in humans and chimpanzees. In the current study, we report measurements of corpus callosum cross-sectional area from midsagittal MR images collected in vivo from 14 adult capuchin monkeys, 9 of which were also characterized for hand preference on a coordinated bimanual task. Adult females were found to have a significantly larger corpus callosum: brain volume ratio, rostral body, posterior midbody, isthmus, and splenium than adult males. Left-handed individuals had a larger relative overall corpus callosum area than did right-handed individuals. Additionally, a significant sex and handedness interaction was found for anterior midbody, with right-handed males having a significantly smaller area than right-handed females. These results suggest that sex and handedness influences on corpus callosum morphology are not restricted to Homo sapiens.     

Rnd2 differentially regulates oligodendrocyte myelination at different developmental periods

In the CNS, oligodendrocyte precursor cells differentiate into oligodendrocytes to wrap their plasma membranes around neuronal axons, generating mature neural networks with myelin sheaths according to spatial and temporal patterns. While myelination is known to be one of the most dynamic cell morphological changes, the overall intrinsic and extrinsic molecular cues controlling myelination remain to be fully clarified. Here, we describe the biphasic roles of Rnd2, an atypical branch of the Rho family GTPase, in oligodendrocyte myelination during development and after maturation in mice. Compared with littermate controls, oligodendrocyte-specific Rnd2 knockout mice exhibit decreased myelin thickness at the onset of myelination but increased myelin thickness in the later period. Larger proportions of Rho kinase and its substrate Mbs, the signaling unit that negatively regulates oligodendrocyte myelination, are phosphorylated at the onset of myelination, while their smaller proportions are phosphorylated in the later period. In addition, we confirm the biphasic role of Rnd2 through experiments with oligodendrocyte-specific Rnd2 transgenic mice. We conclude that Rnd2 positively regulates myelination in the early myelinating period and negatively regulates myelination in the later period. This unique modulator thus plays different roles depending on the myelination period.     

Behavioral asymmetry in BALB/cCF mice with corpus callosum abnormalities]

The possible homology between anomalies of the corpus callosum in mice and in humans remains questionable. The small number of existing behavioural studies in mice have not shown effects, at the behavioural level, of the absence or reduction in size of the corpus callosum. We therefore examined the development, during the first 3 weeks of postnatal life, of a number of simple reflex and integrative responses in the members of a sample of 101 BALB/cCF mice; at autopsy carried out at 50 days of age, 29 mice showed anomalies of the corpus callosum (total absence or an area less than 0.75 mm2). Asymmetry of the development of these responses was measured either as the proportion of animals showing asymmetrical appearance of responses on the left and right sides, and as the delay between appearances on the left and right sides in "asymmetrical" mice. Both measures decreased over the first 15 postnatal days, at the same rate in normal and abnormal mice; in each case the decrease is better described by a second-order, quadratic, function than by a simple linear function. We therefore conclude that the integrity of the corpus callosum is not necessary for the normal maturation of sensorimotor behaviour in mice, and suggest that this conclusion may possibly be explained by the relatively recent appearance of mice in the mammalian radiation.     

Histone modifications affect timing of oligodendrocyte progenitor differentiation in the developing rat brain

Timely differentiation of progenitor cells is critical for development. In this study we asked whether global epigenetic mechanisms regulate timing of progenitor cell differentiation into myelin-forming oligodendrocytes in vivo. Histone deacetylation was essential during a specific temporal window of development and was dependent on the enzymatic activity of histone deacetylases, whose expression was detected in the developing corpus callosum. During the first 10 postnatal days, administration of valproic acid (VPA), the specific inhibitor for histone deacetylase activity, resulted in significant hypomyelination with delayed expression of late differentiation markers and retained expression of progenitor markers. Differentiation resumed in VPA-injected rats if a recovery period was allowed. Administration of VPA after myelination onset had no effect on myelin gene expression and was consistent with changes of nucleosomal histones from reversible deacetylation to more stable methylation and chromatin compaction. Together, these data identify global modifications of nucleosomal histones critical for timing of oligodendrocyte differentiation and myelination in the developing corpus callosum.     

The cortical EEG in the acallosal mouse

The congenital absence of the corpus callosum has recently been found to occur among some mice of the ddN strain in our laboratory. Morphologically, the absence of corpus callosum was classified into two types. One was complete agenesis of corpus callosum, and the other revealed partial agenesis (genu or splenium, or both). In this experiment, the differences of cortical EEG spectral characteristics were studied on the normal, hypogenesis and acallosal mice. A total of 75 male and female adult mice were used. Under light ether anesthesia, five stainless steel electrodes (0.29 mm) were implanted stereotaxically in the bilateral cerebral cortex and cerebellum at a depth of 1.0 mm. After one week recovery period, the correlations of EEG of five combinations, such as homotopic anterior vs. posterior, left vs. right hemispheres, crossed contralateral, and homotopic anterior and posterior to the reference electrode, were analysed with a signal processor. In the complete acallosal mice (n = 11), the correlation coefficients were significantly lower (p less than 0.05), in the crossed contralateral, left vs. right hemispheres, and homotopic posterior to reference. In the hypogenesis of corpus callosum (n = 7), however, they were no significant differences in the correlation compared with normal mice (n = 57). At the end of experiment, the brain was removed and fixed with 10% formalin, then it was cut in half along the midsagittal plane. The midsagittal section was used to examine an outline of the corpus callosum.     

Erythropoietin treatment alleviates ultrastructural myelin changes induced by murine cerebral malaria

ABSTRACT: BACKGROUND: Cerebral malaria (CM) is a severe complication of malaria with considerable mortality. In addition to acute encephalopathy, survivors frequently suffer from neurological sequelae. The pathogenesis is incompletely understood, hampering the development of an effective, adjunctive therapy, which is not available at present. Previously, erythropoietin (EPO) was reported to significantly improve the survival and outcome in a murine CM model. The study objectives were to assess myelin thickness and ultrastructural morphology in the corpus callosum in murine CM and to adress the effects of EPO treatment in this context. METHODS: The study consisted of two groups of Plasmodium berghei-infected mice and two groups of uninfected controls that were either treated with EPO or placebo (n = 4 mice/group). In the terminal phase of murine CM the brains were removed and processed for electron microscopy. Myelin sheaths in the corpus callosum were analysed with transmission electron microscopy and stereology. RESULTS: The infection caused clinical CM, which was counteracted by EPO. The total number of myelinated axons was identical in the four groups and mice with CM did not have reduced mean thickness of the myelin sheaths. Instead, CM mice had significantly increased numbers of abnormal myelin sheaths, whereas EPO-treated mice were indistinguishable from uninfected mice. Furthermore, mice with CM had frequent and severe axonal injury, pseudopodic endothelial cells, perivascular oedemas and intracerebral haemorrhages. CONCLUSIONS: EPO treatment reduced clinical signs of CM and reduced cerebral pathology. Murine CM does not reduce the general thickness of myelin sheaths in the corpus callosum.     

Toll-Like Receptor 2-Mediated Glial Cell Activation in a Mouse Model of Cuprizone-Induced Demyelination

Multiple sclerosis (MS) is a chronic degenerative disease of the central nervous system that is characterized by myelin abnormalities, oligodendrocyte pathology, and concomitant glia activation. The factors triggering gliosis and demyelination are currently not well characterized. New findings suggest an important role of the innate immune response in the initiation and progression of active demyelinating lesions. Especially during progressive disease, aberrant glia activation rather than the invasion of peripheral immune cells is accountable for progressive neuronal injury. The innate immune response can be induced by pathogen-associated or danger-associated molecular patterns, which are identified by pattern recognition receptors (PRRs), including the Toll-like receptors (TLRs). In this study, we used the cuprizone model in mice to investigate the expression of TLR2 during the course of cuprizone-induced demyelination. In addition, we used TLR2-deficient mice to analyze the functional role of TLR2 activation during cuprizone-induced demyelination and reactive gliosis. We show a significantly increased expression of TLR2 in the corpus callosum and hippocampus of cuprizone-intoxicated mice. The absence of receptor signaling in TLR2-deficient mice resulted in less severe reactive astrogliosis in the corpus callosum and cortex. In addition, microglia activation was ameliorated in the corpus callosum of TLR2-deficient mice, but augmented in the cortex compared to wild-type littermates. Extent of demyelination and loss of mature oligodendrocytes was comparable in both genotypes. These results suggest that the TLR2 orchestrates glia activation during gray and white matter demyelination in the presence of an intact blood-brain barrier. Future studies now have to address the underlying mechanisms of the region-specific TLR2-mediated glia activation.     

Intranasal HB-EGF administration favors adult SVZ cell mobilization to demyelinated lesions in mouse corpus callosum

In the adult rodent brain, the subventricular zone (SVZ) represents a special niche for neural stem cells; these cells proliferate and generate neural progenitors. Most of these migrate along the rostral migratory stream to the olfactory bulb, where they differentiate into interneurons. SVZ-derived progenitors can also be recruited spontaneously to damaged brain areas to replace lost cells, including oligodendrocytes in demyelinated lesions. In this study, we searched for factors able to enhance this spontaneous recruitment of endogenous progenitors. Previous studies have suggested that epidermal growth factor (EGF) could stimulate proliferation, migration, and glial differentiation of SVZ progenitors. In the present study we examined EGF influence on endogenous SVZ cell participation to brain repair in the context of demyelinated lesions. We induced a focal demyelinated lesion in the corpus callosum by lysolecithin injection and showed that intranasal heparin-binding epidermal growth factor (HB-EGF) administration induces a significant increase in SVZ cell proliferation together with a stronger SVZ cell mobilization toward the lesions. Besides, HB-EGF causes a shift of SVZ-derived progenitor cell differentiation toward the astrocytic lineage. However, due to the threefold increase in cell recruitment by EGF treatment, the absolute number of SVZ-derived oligodendrocytes in the lesion of treated mice is higher than in controls. These results suggest that enhancing SVZ cell proliferation could be part of future strategies to promote SVZ progenitor cell mobilization toward brain lesions.     

Microstructural White Matter Changes in the Corpus Callosum of Young People with Bipolar Disorder: A Diffusion Tensor Imaging Study

To date, most studies of white matter changes in Bipolar Disorder (BD) have been conducted in older subjects and with well-established disorders. Studies of young people who are closer to their illness onset may help to identify core neurobiological characteristics and separate these from consequences of repeated illness episodes or prolonged treatment. Diffusion tensor imaging (DTI) was used to examine white matter microstructural changes in 58 young patients with BD (mean age 23 years; range 16–30 years) and 40 controls. Whole brain voxelwise measures of fractional anisotropy (FA), parallel diffusivity (λ//) and radial diffusivity (λ⊥) were calculated for all subjects. White matter microstructure differences (decreased FA corrected p<.05) were found between the patients with BD and controls in the genu, body and splenium of the corpus callosum as well as the superior and anterior corona radiata. In addition, significantly increased radial diffusivity (p<.01) was found in the BD group. Neuroimaging studies of young patients with BD may help to clarify neurodevelopmental aspects of the illness and for identifying biomarkers of disease onset and progression. Our findings provide evidence of microstructural white matter changes early in the course of illness within the corpus callosum and the nature of these changes suggest they are associated with abnormalities in the myelination of axons.     

Prefrontal cortex NG2 glia undergo a developmental switch in their responsiveness to exercise

Aerobic exercise is known to influence brain function, e.g., enhancing executive function in both children and adults, with many of these influences being attributed to alterations in neurogenesis and neuronal function. Yet oligodendroglia in adult brains have also been reported to be highly responsive to exercise, including in the prefrontal cortex (PFC), a late myelinating region implicated in working memory. However, whether exercise affects oligodendroglia or myelination in juveniles, either in the PFC or in other brain regions, remains unknown. To address this, both juvenile and young adult mice were provided free access to running wheels for four weeks followed by an analysis of oligodendrocyte development and myelination in the PFC and the corpus callosum, a major white matter tract. Working memory and PFC NG2+ cell development were both affected by exercise in juvenile mice, yet surprisingly these exercise‐mediated effects were distinct in juveniles and young adults. In the PFC, NG2+ cell proliferation was increased in exercising juveniles, but not young adults, whereas newly‐born oligodendrocyte production was increased in exercising young adults, but not juveniles. Although no overall changes in myelin genes were found, elevated levels of Monocarboxylate Transporter 1, a glial lactate transporter important during active myelination, were found in the PFC of exercising young adults. Overall our findings reveal that long‐term exercise modulates PFC glial development and does so differentially in juvenile and young adult mice, providing insight into the cellular responses that may underlie cognitive benefits to teenagers and young adults in response to exercise. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 687–700, 2018     

Only a subset of 12-O-tetradecanoylphorbol-13-acetate-promoted mouse skin papillomas are promotable by benzoyl peroxide

The two-stage skin carcinogenesis model of initiation and promotion in Carcinogenesis-susceptible (Car-S) mice has been used to investigate the pathways of promotional activity of 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbol ester tumor promoter, and benzoyl peroxide (BzPo), a free radical-generating compound. To test whether distinct populations of 9,10-dimethyl-1,2-benzanthracene (DMBA)-initiated epidermal keratinocytes are responsive to the two promoters, tandem experiments were performed. DMBA-initiated Car-S mice were promoted twice weekly with maximal promoting doses of TPA or BzPo. When the number of papillomas/mouse reached a plateau, promotion in the TPA and BzPo groups was switched to BzPo or TPA, respectively, until achievement of a new plateau. Mice promoted with BzPo developed 11.0 +/- 1.3 papillomas/mouse and subsequent TPA promotion induced 13.8 additional papillomas, for a total of 24.8 +/- 2.1 papillomas/mouse. TPA-promoted mice developed 23.3 +/- 1.1 papillomas/mouse, and subsequent BzPo promotion for 91 days did not promote additional papillomas. Our results show a less than additive tumor response after sequential promotion with BzPo and TPA, or vice versa, indicating that the pathways of promotional activity of TPA and BzPo are interacting. While the final papilloma yield was similar at the end of the two tandem promotion experiments independently of promoter sequence, the percentage of mice developing carcinomas was significantly higher in mice that were promoted with BzPo in the first stage. No significant differences in the frequency and type of c-Ha-ras mutations were observed in TPA- and BzPo-promoted tumors, suggesting that promotion of DMBA-initiated cells by BzPo requires introduction of additional molecular alterations compared to TPA.