Neurogenesis in the neonatal rat hippocampus is regulated by sexually dimorphic epigenetic modifiers.

BACKGROUND: Neurogenesis in the hippocampus endures across the lifespan but is particularly prolific during the first postnatal week in the developing rodent brain. The majority of new born neurons are in the dentate gyrus (DG). The number of new neurons born during the first postnatal week in the DG of male rat pups is about double the number in females. In other systems, the rate of cell proliferation is controlled by epigenetic modifications in stem cells. We, therefore, explored the potential impact of DNA methylation and histone acetylation on cell genesis in the developing DG of male and female rats. METHODS: Cell genesis was assessed by quantification of BrdU + cells in the DG of neonatal rats following injections on multiple days. Methylation and acetylation were manipulated pharmacologically by injection of well vetted drugs. DNA methylation, histone acetylation and associated enzyme activity were measured using commercially available colorimetric assays. mRNA was quantified by PCR. Multiple group comparisons were made by one- or two-way ANOVA followed by post-hoc tests controlling for multiple comparisons. Two groups were compared by t test. RESULTS: We found higher levels of DNA methylation in male DG and treatment with the DNA methylating enzyme inhibitor zebularine reduced the methylation and correspondingly reduced cell genesis. The same treatment had no impact on either measure in females. By contrast, treatment with a histone deacetylase inhibitor, trichostatin-A, increased histone acetylation in the DG of both sexes but increased cell genesis only in females. Females had higher baseline histone deacetylase activity and greater inhibition in response to trichostatin-A treatment. The mRNA levels of the proproliferative gene brain-derived neurotrophic factor were greater in males and reduced by inhibiting both DNA methylation and histone deacetylation only in males. CONCLUSIONS: These data reveal a sexually dimorphic epigenetically based regulation of neurogenesis in the DG but the mechanisms establishing the distinct regulation involving DNA methylation in males and histone acetylation in females is unknown.

Squamous cell hyperplastic foci: precursors of cutaneous papillomas induced in SENCAR mice by a two-stage carcinogenesis regimen.

We have conducted a series of experiments to characterize the lesions that are precursors of cutaneous papillomas in SENCAR mice initiated with 7,12-dimethylbenz(a)anthracene (DMBA) and promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). The first grossly detectable lesions at sites where papillomas subsequently developed were papules, slightly raised areas of skin ranging in diameter from 0.25 to approximately 1.5 mm. Papules were first detected in DMBA-initiated mice 21 days after the start of dosing with TPA. Of 78 DMBA/TPA-induced papules tracked during 15 weeks of TPA treatments, 68% progressed to papillomas, 9% persisted as papules, and 22% completely regressed. Histological evaluation of serial sections of 69 DMBA/TPA-induced papules revealed that they were focal hyperplastic lesions that we refer to as squamous cell hyperplastic foci (SCHF). These hyperproliferative lesions appeared to progress through two distinct stages. Stage I SCHF were characterized as regular hyperplastic foci involving the interfollicular epidermis and the outer root sheaths of 1 or more hair follicles down to the level of the sebaceous glands. Stage II SCHF were foci of irregular epithelial hyperplasia with increased fibrovascular stroma and involved from 3 to >10 hair follicles. Prominent dilated capillaries and inflammatory cell infiltrates were frequently associated with both stage I and II SCHF. Ha-ras gene codon 61 mutations were detected in 7 of 10 stage I SCHF and 13 of 14 stage II SCHF microdissected from histological sections and 7 of 7 of whole papules by mutation-specific PCR analysis. These data provide molecular evidence that SCHF are foci of initiated cells. Further study of these lesions may contribute to more fully defining the sequence of molecular and cellular changes necessary for tumorigenesis in mouse skin. SCHF may also have utility as early indicators of potential skin tumorigenicity in cancer bioassays.

Evidence that initiated keratinocytes clonally expand into multiple existing hair follicles during papilloma histogenesis in SENCAR mouse skin.

We have previously shown that the precursors of cutaneous papillomas in SENCAR mice initiated with 7,12-dimethylbenz[a]anthracene and promoted with 12-O-tetradecanoylphorbol-13-acetate are focal hyperplastic lesions that we refer to as squamous cell hyperplastic foci (SCHF). Ha-ras gene codon 61 mutations were frequently found in SCHF, providing evidence that these lesions represent clones of initiated cells. We report here the pathogenesis of multiple hair follicle involvement in more advanced SCHF and describe the role of the hair follicle in papilloma histogenesis. Detailed histological evaluation of 83 SCHF and 25 early papillomas revealed a morphological continuum from the least developed SCHF, involving only one hair follicle, to advanced SCHF and early papillomas, which involved more than 10 hair follicles. These results provide evidence of the recruitment of additional hair follicles as SCHF progress. In advanced SCHF and early papillomas the bulk of the epithelial component in all cases consisted of several markedly hyperplastic adjacent hair follicles, whereas the involved interfollicular epidermis (IFE) was generally less hyperplastic. All of the hair follicles involved in SCHF appeared to have been preexisting, based on their pattern of spacing, that they were consistently normal appearing below the level of the sebaceous glands, and that they were in the same phase of the hair cycle as surrounding, uninvolved hair follicles. Also, no evidence of follicular neogenesis was observed in serially sectioned SCHF, and coalescence of smaller lesions was rare. To investigate whether the involvement of multiple hair follicles in SCHF was due to expansion of initiated cells into existing hair follicles or, possibly, to a paracrine mechanism, we analyzed different levels of three serially sectioned SCHF and one early papilloma for Ha-ras mutations. These analyses revealed cells with Ha-ras gene codon 61 mutations at multiple levels that involved different hair follicles. Overall, our results provide evidence that as initiated cells clonally expand, they spread across the IFE and populate the upper permanent portions of existing hair follicles. The abnormal proliferation of the infundibula of the hair follicles involved in SCHF appears to give rise to most of the epithelial component of papillomas.

Transforming growth factor-beta 1 expression in Syrian hamster cheek pouch carcinogenesis.

The expression pattern of transforming growth factor-beta 1 (TGF-beta 1) during the stages of complete carcinogenesis in the hamster cheek pouch model was studied. The right cheek pouches of 18 male hamsters were treated with 0.5%, 7,12-dimethylbenz[a]anthracene (DMBA) for 16 wk. TGF-beta 1 was detected immunohistochemically in the resulting samples with two different polyclonal monospecific antibodies that recognize intracellular and extracellular forms of TGF-beta 1. In the normal cheek pouch, extracellular protein stained the corium strongly, but the reaction was not evenly distributed. As treatment progressed, the reaction increased in both area and intensity; the peak was reached at 8 wk. Intracellular TGF-beta 1 expression followed a similar pattern, with a peak at 4 wk of treatment. The results of northern blot analysis were concordant with the immunohistochemical results. Overexpression of TGF-beta 1 was also observed in the malignant tumors, but only the extracellular form of the protein was present; intracellular TGF-beta 1 was not detected in these tumors. The expression of TGF-beta 1 in this carcinogenesis model seems to have two formal stages, the first being an overexpression step as a reaction to the uncontrolled growth and the second being one in which tumors have no internal expression of TGF-beta 1 but in which external protein accumulates in the surrounding stroma. A possible explanation of this paradox may be that TGF-beta 1 has functions other than its growth-repressing activity.

Activating mutation of the Ha-ras gene in chemically induced tumors of the hamster cheek pouch.

The presence of an activating mutation in the Ha-ras gene in hamster cheek pouch tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) complete carcinogenesis was investigated. The normal sequence of a fragment of genomic DNA encompassing codon 61 of the Ha-ras gene was amplified by the polymerase chain reaction using primers designed for a highly conserved region of the mouse Ha-ras-1 gene. The sequence of the amplified fragment was determined by a direct sequencing technique and exhibited 83.3% and 87.5% homology with the corresponding human and mouse sequences, respectively. At the amino acid level, the sequence was identical among the three species. Paraffin sections of 11 squamous cell carcinomas of the cheek pouch were used to detect mutated Ha-ras alleles. DNA sequencing of the tumors showed that six of 11 tumors presented an A----T transversion in the second position of codon 61, resulting in an amino acid change from glycine to leucine. As has been demonstrated in other systems, we have shown a specific mutation of the Ha-ras gene in chemically induced tumors of the hamster cheek pouch, further supporting the role of this oncogene in chemical carcinogenesis.

Quantitation of HeLa cell monolayer invasion by Shigella and Salmonella species.

A major determinant in the virulence of Salmonella and Shigella spp. is the ability of these organisms to invade epithelial cells of the gastrointestinal mucosa and multiply intracellularly. The invasion of cell culture monolayers is a convenient experimental system to evaluate eucaryotic cell penetration and is correlated with the potential of a strain to cause human disease. We have developed an agarose-L agar overlay technique which allows for the convenient quantitation of the number of infected tissue culture cells in a monolayer. Bacterial strains were introduced onto antibiotic-free HeLa cell monolayers. Infected monolayers were washed, and noninternalized bacteria were counterselected with kanamycin (50 micrograms/ml). The number of infected HeLa cells present was determined by overlaying the monolayer with distilled water-agarose (0.5 to 1.5%) followed by an equal volume of 2X L agar. Bacterial colonies formed over infected cells in 24 h at 37 degrees C, and wells were counted with a dissecting microscope under X2 power. Bacterial colonies were not observed with noninvasive variants of Shigella spp. To obtain countable wells (20 to 200 CFU) the multiplicity of infection or invasion times were adjusted. With a 90-min invasion time, the invasive potential of a strain was reflected by the multiplicity of infection needed to produce countable wells. The quantitation of bacterium-invaded cells by using standard bacteriological methods is a convenient and rapid method to evaluate the invasive potential of bacterial strains. Additionally, parameters essential for the invasive process can easily be investigated.