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(Eaf2) is a potential human tumor suppressor that exhibits frequent allelic loss and downregulation in high-grade prostate cancer. U19/Eaf2, along with its homolog Eaf1, has been reported to regulate transcriptional elongation via interaction with the eleven-nineteen lysine-rich leukemia (ELL) family of proteins. To further explore the tumor-suppressive effects of U19/Eaf2, we constructed and characterized a murine U19/Eaf2-knockout model. Homozygous or heterozygous deletion of U19/Eaf2 resulted in high rates of lung adenocarcinoma, B-cell lymphoma, hepatocellular carcinoma and prostate intraepithelial neoplasia. Within the mouse prostate, U19/Eaf2 deficiency enhanced cell proliferation and increased epithelial cell size. The knockout mice also exhibited cardiac cell hypertrophy. These data indicate a role for U19/Eaf2 in growth suppression and cell size control as well as argue for U19/Eaf2 as a novel tumor suppressor in multiple mouse tissues. The U19/Eaf2 knockout mouse also provides a unique animal model for three important cancers: lung adenocarcinoma, B-cell lymphoma and hepatocellular carcinoma.

converges on the central premise that maximal immunotherapeutic efficacy in subjects with advanced cancer requires both IDO enzyme-and non-enzyme-neutralization, which is not adequately addressed by available IDO-targeting pharmacologic approaches at this time.

BackgroundThe C-terminal Eps15 homology domain-containing protein 1 (EHD1) is ubiquitously expressed and regulates the endocytic trafficking and recycling of membrane components and several transmembrane receptors. To elucidate the function of EHD1 in mammalian development, we generated Ehd1-/- mice using a Cre/loxP system.ResultsBoth male and female Ehd1-/- mice survived at sub-Mendelian ratios. A proportion of Ehd1-/- mice were viable and showed smaller size at birth, which continued into adulthood. Ehd1-/- adult males were infertile and displayed decreased testis size, whereas Ehd1-/- females were fertile. In situ hybridization and immunohistochemistry of developing wildtype mouse testes revealed EHD1 expression in most cells of the seminiferous epithelia. Histopathology revealed abnormal spermatogenesis in the seminiferous tubules and the absence of mature spermatozoa in the epididymides of Ehd1-/- males. Seminiferous tubules showed disruption of the normal spermatogenic cycle with abnormal acrosomal development on round spermatids, clumping of acrosomes, misaligned spermatids and the absence of normal elongated spermatids in Ehd1-/- males. Light and electron microscopy analyses indicated that elongated spermatids were abnormally phagocytosed by Sertoli cells in Ehd1-/- mice.ConclusionsContrary to a previous report, these results demonstrate an important role for EHD1 in pre- and post-natal development with a specific role in spermatogenesis.

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The mammalian genome encodes a family of lactate dehydrogenase (LDH) isozymes. Two of these, ldha and ldhb, are expressed ubiquitously. The ldhc gene is active only in the germinal epithelium during spermatogenesis. In our analysis of ldhc gene regulation, we found that a 60-base pair promoter sequence was sufficient for testis-specific expression in an in vitro transcription assay. To confirm these findings, a genomic fragment containing 100 base pairs overlapping the transcription start site was isolated and linked to the Escherichia coli lacZ gene. We report that this genomic fragment drives testis-specific expression in transgenic mice. We conclude that transcription of the transgene and possibly of the endogenous ldhc gene is restricted to leptotene/ pachytene primary spermatocytes.Spermatogenesis is a complex process requiring the coordinate expression of a number of genes. One of these, ldhc, encodes the testis-specific isozyme of lactate dehydrogenase (LDH-C 4 ). 1 The kinetic properties and enzyme-substrate interactions (1) suggest that LDH-C 4 is uniquely suited for satisfying the metabolic requirements of differentiating germ cells and functional spermatozoa. Ldhc is one of several genes that are expressed at various stages of spermatogenesis and either encode unique proteins or produce mRNAs specific to male germ cells (2). A clone containing the 5Ј-flanking region of the murine ldhc gene was isolated from a mouse genomic library. There are no permanent cell lines available for promoter analysis in male germ cells, and reliable methods for transfecting primary germ cells have not been developed. We have been able to circumvent this problem successfully with an in vitro transcription assay. Promoter activity was demonstrated within a 720-bp fragment by in vitro transcription assays in testis nuclear extract. Liver nuclear extracts significantly repressed ldhc promoter activity in this assay system. Analysis of a series of deletion mutants revealed that a 60-bp core promoter sequence was sufficient to direct basal, testis-specific transcription (3). To confirm these findings, a genomic fragment containing approximately 100 bp immediately upstream from the transcription start site was isolated and linked to the Escherichia coli lacZ reporter gene. We report that this genomic fragment drives testis-specific ␤-galactosidase expression in transgenic mice. We conclude that transcription of the transgene is restricted to leptotene-pachytene-stage primary spermatocytes, even though endogenous LDH-C 4 increases in concentration during spermatogenesis. EXPERIMENTAL PROCEDURESGeneration of Transgenic Mice-Two constructs containing murine ldhc promoter sequences were designed to direct expression of lacZ. A 720-bp mouse ldhc fragment (Ϫ710 to ϩ12) for construct 100H␤ was amplified by PCR as described previously (3). Similarly, the 700-bp fragment for construct 100⌬H␤ was amplified as described in Zhou and Goldberg (4). These two PCR products were subcloned into the blunted EcoRI site of the pNAss␤ vector from CLO...

The zinc finger transcription factor GLI1 is the mediator of signaling by members of the Hedgehog (Hh) family. Male mice in which Desert hedgehog (Dhh), an Hh homologue expressed in Sertoli cells of the testis, was knocked out are sterile, suggesting that the Dhh/GLI1 pathway plays a role in spermatogenesis. Using an antiserum raised against human GLI1, we found that during the first round of spermatogenesis, GLI1 expression is initially cytoplasmic, then shifts to the nuclei of Sertoli and germ cells, and finally shifts back to the cytoplasm. In the adult mouse testis, GLI1 expression localized to the nuclei of germ cells, beginning with pachytene cells and persisting through round spermatids. Localization of GLI1 in elongating spermatids shifted from the nucleus to the cytoplasm and became associated with microtubules. We also examined a line of transgenic mice that overexpressed human GLI1. Male mice in this line were sterile. Spermatogenesis was blocked at the pachytene stage, and a subset of the morphologically indistinguishable pachytene cells underwent apoptosis. Patched-2, which is a Dhh receptor, and Fused, another component of the signal transduction pathway, are expressed in Leydig cells and in primary and secondary spermatocytes. Expression of GLI1 in the same cell types as Patched-2 and Fused and the disruption of spermatogenesis by GLI1 overexpression suggest that GLI1 is the mediator of the Dhh signal in the testis, and that it may be a regulator of spermatogenesis.

SummaryTransgenic mice with a y2b transgene were produced to investigate whether y2b can replace P in the development of B lymphocytes . Transgenic y2b is present on the surface of B cells. Young transgenic mice have a dramatic decrease in B cell numbers, however, older mice have almost normal B cell numbers . Strikingly, all y2b-expressing B cells in the spleen also express h . The same is true for mice with a hybrid transgene in which the A. transmembrane and intracytoplasmic sequences replace those of y2b (72b-,umem) . The B cell defect is not due to toxicity of y2b since crosses between y2b transgenic and A transgenic mice have normal numbers of B cells. Presence of the y2b transgene strongly enhances the feedback inhibition of endogenous heavy chain gene rearrangement. Light chain genes are expressed normally, and the early expression of transgenic light chains does not improve B cell maturation. When the endogenous A locus is inactivated, B cells do not develop at all in y2b transgenic mice. The data suggest that y2b cannot replace A in promoting the developmental maturation of B cells, but that it can cause feedback inhibition ofheavy chain gene rearrangement . Thus, the signals for heavy chain feedback and B cell maturation appear to be different .