Expression of genes controlling steroid metabolism and action in granulosa-lutein cells of women with polycystic ovaries.

INTRODUCTION: Aberrant function of granulosa cells has been implicated in the pathophysiology of PCOS. MATERIALS & METHODS: Granulosa lutein (GL) cells were collected during oocyte retrieval for IVF/ICSI. RT-qPCR was used to compare gene expression between 12 control women, 12 with ovulatory PCO and 12 with anovulatory PCOS. To examine which genes are directly regulated by androgens, GL cells from an additional 12 control women were treated in-vitro with 10 nM dihydrotestosterone (DHT). RESULTS: GL cells from women with PCOS showed reduced expression of CYP11A1 3-fold (p = 0.005), HSD17B1 1.8-fold (p = 0.02) and increased expression of SULT1E1 7-fold (p = 0.0003). Similar results were seen in ovulatory women with PCO. GL cells treated with 10 nM DHT showed a 4-fold (p = 0.03) increase in expression of SULT1E1 and a 5-fold reduction in SRD5A1 (p = 0.03). CONCLUSIONS: These findings support the notion that aberrant regulation of steroid metabolism or action play a part in ovarian dysfunction in PCOS.

Abnormal rearrangement within the alpha/delta T-cell receptor locus in lymphomas from Atm-deficient mice.

Atm-deficient mice (Atm(-/-)) recapitulate many aspects of the ataxia telangiectasia (AT) syndrome, including the susceptibility to tumors of lymphoid origin. To investigate the mechanism of tumorigenesis, we have examined a panel of 8 thymic lymphomas from Atm(-/-) mice. All Atm(-/-) tumors are of thymic lymphoblastoid origin, display an immature CD3(-) and CD4(+)/CD8(+) phenotype, and arise coincident with V(D)J recombination. Cytogenetically, all tumors are diploid or near diploid but exhibit multiple chromosome aberrations with an average of 4 abnormal chromosomes per tumor. All the tumors revealed chromosome 14 rearrangements precisely at the T-cell receptoralpha/delta (Tcralpha/delta) locus, suggesting the involvement of V(D)J recombination in these translocations. In addition, 11.5% of Atm(-/-) peripheral T cells showed chromosome 14 translocations, suggesting that rearrangements at the Tcralpha/delta locus occur early during tumor development in the absence of ATM. However, additional genetic aberrations are required for tumorigenesis. For example, translocations involving chromosome 12, often with chromosome 14 (more than 60%), and partial or complete trisomy of chromosome 15, with copy number increases of the c-myc oncogene were frequently observed. These observations suggest that ATM is required for normal rearrangement of the Tcralpha/delta locus but not for V(D)J recombination at other loci. The mechanisms that lead to tumorigenesis may be due to the involvement of ATM in monitoring double-stranded DNA breaks. (Blood. 2000;96:1940-1946)

Nucleoli in a pronuclei-stage mouse embryo are represented by major satellite DNA of interconnecting chromosomes.

OBJECTIVE: To investigate the arrangement of chromosomes within pronuclei-stage mouse zygotes. DESIGN: In vitro study. SETTING: Academic medical center. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Location of major alpha-satellite DNA, centromeres, and telomeres, and relative location of chromosomes. RESULT(S): Chromosomes appeared to be oriented inward by centromeres and to be interconnected by major alpha-satellite DNA, which appeared to be the sole DNA component of the nucleoli. This chromosomal arrangement persisted throughout interphase. Chromosomal painting failed to identify chromosomal ordering within pronuclei. CONCLUSION(S): Pronuclear nucleoli are represented by alpha-satellite sequences of interconnecting chromosomes that hold all chromosomes together during interphase. Chromosomes within the pronucleus are randomly positioned relative to each other.

A recurring pattern of chromosomal aberrations in mammary gland tumors of MMTV-cmyc transgenic mice.

Mice carrying the MMTV-cmyc transgene develop mammary tumors at 9 to 12 months of age. Little is known about karyotypic changes in this model of human breast cancer. We have developed and applied molecular cytogenetic techniques to study chromosomal aberrations that occur in these tumors, namely, comparative genomic hybridization and spectral karyotyping. Cell lines from eight tumors were established and analyzed, four of which carried a heterozygous p53 mutation. All of the tumor cell lines revealed increases in ploidy and/or multiple numerical and structural chromosomal aberrations. No consistent differences were observed between cmyc/p53+/+ and cmyc/p53+/- tumors, suggesting that cmyc induces karyotype instability independent of p53 status. Loss of whole chromosome (Chr) 4 was detected in five of the eight tumors. Parts of Chr 4 are syntenic to human 1p31-p36, a region that is also deleted in human breast carcinomas. Four tumors carried translocations involving the distal portion of Chr 11 (syntenic to human chromosome arm 17q), including two translocations T(X;11), with cytogenetically identical breakpoints. We compare the pattern of chromosomal aberrations with human breast cancers, find similarities in several syntenic regions, and discuss the potential of an interspecies cytogenetic map of chromosomal gains and losses.

Amplification of Ki-ras and elevation of MAP kinase activity during mammary tumor progression in C3(1)/SV40 Tag transgenic mice.

We have previously documented that transgenic mice expressing SV40 Tag regulated by the rat prostatic steroid-binding protein C3(1) 5'-flanking region display multistage mammary tumorigenesis. To delineate genetic changes associated with mammary tumor progression, comparative genomic hybridization (CGH) was performed. CGH revealed a consistent gain of the telomeric region of chromosome 6. This region contains the Ki-ras proto-oncogene. Analyses of genomic DNA by Southern blot demonstrated up to 40-fold amplification of the Ki-ras gene. Ki-ras amplification was detected in 12, 46 and 68% of tumors from 4, 5 and 6 month old mice, respectively, whereas no amplifications were found in any preneoplastic mammary tissues. Tumors bearing Ki-ras gene amplification exhibited high levels of Ki-ras RNA and protein. The over-expressed Ki-Ras protein in these tumors appeared functionally active as indicated by the elevated MAP kinase activity. These data demonstrate that while Ki-ras amplification might not be an early event, there is a strong association between Ki-ras amplification and over-expression and mammary tumor progression in this model. This study also shows that CGH is a powerful and useful technique for identifying chromosomal copy number changes during tumor progression, and that this model may provide a predictable in vivo system for studying gene amplification.

Atm deficiency results in severe meiotic disruption as early as leptonema of prophase I.

Infertility is a common feature of the human disorder ataxia-telangiectasia and Atm-deficient mice are completely infertile. To gain further insight into the role of ATM in meiosis, we examined meiotic cells in Atm-deficient mice during development. Spermatocyte degeneration begins between postnatal days 8 and 16.5, soon after entry into prophase I of meiosis, while oocytes degenerate late in embryogenesis prior to dictyate arrest. Using electron microscopy and immunolocalization of meiotic proteins in mutant adult spermatocytes, we found that male and female gametogenesis is severely disrupted in Atm-deficient mice as early as leptonema of prophase I, resulting in apoptotic degeneration. A small number of mutant cells progress into later stages of meiosis, but no cells proceed beyond prophase I. ATR, a protein related to ATM, DMC1, a RAD51 family member, and RAD51 are mislocalized to chromatin and have reduced localization to developing synaptonemal complexes in spermatocytes from Atm-deficient mice, suggesting dysregulation of the orderly progression of meiotic events. ATM protein is normally present at high levels primarily in ova cytoplasm of developing ovarian follicles, and in the nucleus of spermatogonia and to a lesser extent in spermatoctyes, but without localization to the synaptonemal complex. We propose a model in which ATM acts to monitor meiosis by participation in the regulation or surveillance of meiotic progression, similar to its role as a monitor of mitotic cell cycle progression.

Myc/p53 interactions in transgenic mouse mammary development, tumorigenesis and chromosomal instability.

We have examined defects in mammary development and tumorigenesis in a transgenic model expressing the c-myc gene under the MMTV-LTR promoter. The stochastic tumors which arise from hyperplastic ductal and lobular lesions in this model are characterized by high rates both of apoptosis and of chromosomal instability. Since the p53 gene product is thought to be central in the maintenance of genomic integrity, in part due to its ability to induce apoptosis in cells harboring DNA damage, we examined its expression and possible mutation. Initially, we observed that unmutated p53 is strongly expressed in premalignant mammary glands and in mammary tumors derived from the MMTV-c-myc strain. We then mated the MMTV-myc strain to a p53-deficient strain as a means of examining the effect of this lesion on mammary development and tumorigenesis in the context of c-myc overexpression. A lack of both p53 alleles in the presence of c-myc overexpression resulted in a dramatic hyerplastic alteration in mammary gland development. Specifically, in female bitransgenic MMTV-c-myc/p53 null mice (MMTV-myc/p53(-/-)), lobular hyperplasias were observed at almost every ductal end bud as early as 32 days of age. In contrast, only mild ductal and lobular hyperplasias were seen in MMTV-myc mice that contained both p53 alleles (MMTV-myc/p53(+/+)); an intermediate phenotype occurred in mice with a single intact (MMTV-myc/p53(+/-)) p53 allele. Mammary carcinomas arose with a high frequency in MMTV-myc/p53(+/-) mice; the tumors were comparable in frequency, histology and apoptotic index to the tumors in MMTV-myc/p53(+/+) mice. Also, as previously observed (Elson et al., 1995), lymphomas arose with extremely short latency in MMTV-myc/p53(-/-) mice, precluding study of the fate of their hyperplastic mammary lesions in situ. The frequency of p53 mutations in MMTV-myc/p53(+/+) and MMTV-myc/p53(+/-) mammary tumors and in cell lines derived from these tumors was examined by direct sequencing. No point mutations or deletions in p53 were observed in mammary tumors or cell lines from either genotype. Finally, a detailed chromosomal analysis using multicolor spectral karyotyping (SKY) revealed that there were multiple chromosomal alterations in the c-myc-overexpressing cells that contained either one or two unmutated p53 alleles. Variable ploidy changes, a common translocation of chromosome 11, and other chromosomal aberrations were observed. Our data thus support an interaction between c-Myc and p53 in mammary development, but suggest that loss of p53 is required neither for c-myc-dependent tumorigenesis nor for c-myc-dependent chromosomal instability.

Partial rescue of the prophase I defects of Atm-deficient mice by p53 and p21 null alleles.

Patients with the human disorder ataxia-telangiectasia (A-T; refs 1,2) and Atm-deficient mice have a pleiotropic phenotype that includes infertility. Here we demonstrate that male gametogenesis is severely disrupted in Atm-deficient mice in the earliest stages of meiotic prophase I, resulting in apoptotic degeneration. Atm is required for proper assembly of Rad51 onto the chromosomal axial elements during meiosis. In addition, p53, p21 and Bax are elevated in testes from Atm-deficient mice. To determine whether these elevated protein levels are important factors in the meiotic disruption of Atm-deficient mice, we analysed the meiotic phenotype of Atm/p53 or Atm/p21 double mutants. In these double mutants, meiosis progressed to later stages but was only partly rescued. Assembly of Rad51 foci on axial elements remained defective, and gametogenesis proceeded only to pachytene of prophase I. Previous results demonstrated that mice homozygous for a null mutation in Rad51 (ref. 6) display an early embryonic lethal phenotype that can be partly rescued by removing p53 and/or p21. Because Atm-deficient mice are viable but completely infertile, our studies suggest that the Rad51 assembly defects and elevated levels of p53, p21 and Bax represent tissue-specific responses to the absence of Atm.

Tumor cytogenetics revisited: comparative genomic hybridization and spectral karyotyping.

Fluorescence in situ hybridization techniques allow the visualization and localization of DNA target sequences on the chromosomal and cellular level and have evolved as exceedingly valuable tools in basic chromosome research and cytogenetic diagnostics. Recent advances in molecular cytogenetic approaches, namely comparative genomic hybridization and spectral karyotyping, now allow tumor genomes to be surveyed for chromosomal aberrations in a single experiment and permit identification of tumor-specific chromosomal aberrations with unprecedented accuracy. Comparative genomic hybridization utilizes the hybridization of differentially labeled tumor and reference DNA to generate a map of DNA copy number changes in tumor genomes. Comparative genomic hybridization is an ideal tool for analyzing chromosomal imbalances in archived tumor material and for examining possible correlations between these findings and tumor phenotypes. Spectral karyotyping is based on the simultaneous hybridization of differentially labeled chromosome painting probes (24 in human), followed by spectral imaging that allows the unique display of all human (and other species) chromosomes in different colors. Spectral karyotyping greatly facilitates the characterization of numerical and structural chromosomal aberrations, therefore improving karyotype analysis considerably. We review these new molecular cytogenetic concepts, describe applications of comparative genomic hybridization and spectral karyotyping for the visualization of chromosomal aberrations as they relate to human malignancies and animal models thereof, and provide evidence that fluorescence in situ hybridization has developed as a robust and reliable technique which justifies its translation to cytogenetic diagnostics.

Multicolour spectral karyotyping of mouse chromosomes.

Murine models of human carcinogenesis are exceedingly valuable tools to understand genetic mechanisms of neoplastic growth. The identification of recurrent chromosomal rearrangements by cytogenetic techniques serves as an initial screening test for tumour specific aberrations. In murine models of human carcinogenesis, however, karyotype analysis is technically demanding because mouse chromosomes are acrocentric and of similar size. Fluorescence in situ hybridization (FISH) with mouse chromosome specific painting probes can complement conventional banding analysis. Although sensitive and specific, FISH analyses are restricted to the visualization of only a few mouse chromosomes at a time. Here we apply a novel imaging technique that we developed recently for the visualization of human chromosomes to the simultaneous discernment of all mouse chromosomes. The approach is based on spectral imaging to measure chromosome-specific spectra after FISH with differentially labelled mouse chromosome painting probes. Utilizing a combination of Fourier spectroscopy, CCD-imaging and conventional optical microscopy, spectral imaging allows simultaneous measurement of the fluorescence emission spectrum at all sample points. A spectrum-based classification algorithm has been adapted to karyotype mouse chromosomes. We have applied spectral karyotyping (SKY) to chemically induced plasmocytomas, mammary gland tumours from transgenic mice overexpressing the c-myc oncogene and thymomas from mice deficient for the ataxia telangiectasia (Atm) gene. Results from these analyses demonstrate the potential of SKY to identify complex chromosomal aberrations in mouse models of human carcinogenesis.

Atm-deficient mice: a paradigm of ataxia telangiectasia.

A murine model of ataxia telangiectasia was created by disrupting the Atm locus via gene targeting. Mice homozygous for the disrupted Atm allele displayed growth retardation, neurologic dysfunction, male and female infertility secondary to the absence of mature gametes, defects in T lymphocyte maturation, and extreme sensitivity to gamma-irradiation. The majority of animals developed malignant thymic lymphomas between 2 and 4 months of age. Several chromosomal anomalies were detected in one of these tumors. Fibroblasts from these mice grew slowly and exhibited abnormal radiation-induced G1 checkpoint function. Atm-disrupted mice recapitulate the ataxia telangiectasia phenotype in humans, providing a mammalian model in which to study the pathophysiology of this pleiotropic disorder.

Evidence for different signalling pathways of PKC zeta and ras-p21 in Xenopus oocytes.

Considerable effort has been devoted to identifying critical steps in mitogenic signal transduction pathways. Recently, the atypical PKC zeta isoform has attracted great interest since it has been reported to induce GVBD in Xenopus oocytes and transformation of NIH3T3 fibroblasts, two processes closely linked with the regulation of cell division. Furthermore, PKC zeta has been proposed as an essential effector for ras-p21 function and therefore may be an essential component of the signalling pathway(s) activated by mitogens. In this study we have analysed the responses induced in Xenopus oocytes after microinjection of purified recombinant PKC zeta protein. Microinjection of PKC zeta induced the early activation of MPF which precedes GVBD and also induced the activation of MAP kinase and S6 kinase II. The activation of MPF, MAP kinase and S6 kinase II by PKC zeta was sensitive to cycloheximide, while induction of GVBD was independent of protein synthesis. These results indicate that PKC zeta induces the activation of at least two pathways, only one of them leading to the activation of MAP kinase. By contrast, neither the induction of GVBD nor the activation of MPF, MAPK and S6 kinase II induced by the ras-p21 protein were dependent on protein synthesis. Thus, the comparison of these responses suggests that PKC zeta most likely does not mediate the ras-induced signal transduction pathway in Xenopus laevis oocytes.

Different ARF domains are required for the activation of cholera toxin and phospholipase D.

ADP-ribosylation factors (ARFs), initially described as activators of cholera toxin ADP-ribosyltransferase activity, regulate intracellular vesicular membrane trafficking and stimulate a phospholipase D (PLD) isoform. ARF-like (ARL) proteins are structurally related to ARFs but do not activate cholera toxin and have relatively little effect on PLD. A new human ARL gene termed hARL1, which shares 57% amino acid identity with hARF1, was identified using a polymerase chain reaction-based cloning method. To determine whether different structural elements are responsible for the activation structural elements are responsible for the activation of the A subunit of cholera toxin and PLD, chimeric proteins were constructed by switching the amino-terminal 73 amino acids of ARF1 and ARL1. The recombinant rL73/F protein, in which the amino-terminal 73 amino acids of ARL1 replaced those of ARF1, activated the A subunit of cholera toxin, whereas the rF73/L protein, in which the NH2-terminal 73 amino acids of ARF1 replaced those of ARL1, was inactive. The two chimeric proteins had quite opposite effects on PLD activity. rF73/L activated PLD as effectively as rARF1, whereas rL73/F protein activated PLD only slightly. It appears that the amino-terminal region of ARF1 is not critical for its action as a GTP-dependent activator of cholera toxin, whereas it is necessary for activation of the putative effector enzyme, PLD.

Activation of rat brain phospholipase D by ADP-ribosylation factors 1,5, and 6: separation of ADP-ribosylation factor-dependent and oleate-dependent enzymes.

Two major forms of phospholipase D (PLD) activity, solubilized from rat brain membranes with Triton X-100, were separated by HPLC on a heparin-5PW column with buffer containing octyl glucoside. One form was completely dependent on sodium oleate for activity. The other, which was dramatically activated by the addition of ADP-ribosylation factor (ARF) 1 and guanine 5' [gamma-thio]triphosphate, required the presence of phosphatidylinositol 4,5-bisphosphate in the phosphatidylcholine substrate for demonstration of activity, as described by others. Oleate-dependent activity was unaffected by guanine 5' [gamma-thio]triphosphate, or phosphatidylinositol 4,5-bisphosphate. Both sodium oleate-and ARF-dependent activities catalyzed transphosphatidylation, thus identifying them as PLDs. ARF-dependent PLD was activated by recombinant ARF5 (class II) and ARF6 (class III), as well as ARF1 (class I). Myristoylated recombinant ARFs were more effective than their nonmyristoylated counterparts. ARFs were originally identified as activators of cholera toxin ADP-ribosyltransferase activity. The effects of recombinant ARF proteins from the three classes on cholera toxin activity (assayed under conditions identical to those used to assay PLD activity) did not, however, correlate with those on PLD, consistent with the notion that different aspects of ARF structure are involved in the two functions.

Activation of the c-Raf protein kinase by protein kinase C phosphorylation.

The product of the c-raf-1 proto-oncogene is a cytoplasmic serine/threonine protein kinase that appears to be activated in signal transduction from a variety of cell-surface receptors. The mechanism of c-Raf activation upon stimulation of cell-surface receptors is not clear, but there seem to exist multiple pathways of activation which involve tyrosine and/or serine phosphorylation of the c-Raf protein in vivo. The activated state of Raf is reflected in an increased apparent molecular weight of the Raf protein in sodium dodecyl sulfate-polyacrylamide gels owing to hyperphosphorylation. The tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) is one of the agents able to induce this hyperphosphorylation of Raf in vivo, suggesting that protein kinase C (PKC) may be involved in the activation of c-Raf in particular situations. Using recombinant baculoviruses expressing PKC and Raf polypeptides, we show here that conventional PKC types (alpha, beta, gamma) but not novel types (delta, zeta, eta) or the unrelated Mos kinase are able to activate c-Raf in a TPA-dependent manner upon coexpression in insect cells. Direct phosphorylation of the Raf protein with PKC in vitro also enhanced the kinase activity of c-Raf, suggesting that c-Raf acts immediately downstream of PKC in a protein kinase cascade which is triggered by TPA and may lead to transcriptional activation of TPA-inducible genes and tumor promotion.

Protein kinase C group B members PKC-delta, -epsilon, -zeta and PKC-L(eta). Comparison of properties of recombinant proteins in vitro and in vivo.

Of the recently identified protein kinase C (PKC) types of group B (delta, epsilon, zeta, eta, PKC-L), only PKC-epsilon has been characterized in great detail. In order to compare the regulatory and catalytic properties of these new kinases, we have expressed PKC-delta, -epsilon, -zeta and PKC-L as recombinant proteins from their cDNAs in insect cells via baculovirus vectors and in mammalian COS-1 cells. After expression in insect cells, phorbol ester binding and kinase activities of the group B enzymes were compared with the respective activities of a member of group A, PKC-gamma. Although PKC-delta and PKC-L(eta) bind phorbol ester to a similar or the same extent as PKC-gamma, they show a distinctively different behaviour towards conventional PKC substrates such as histone, myelin basic protein, protamine and protamine sulphate, suggesting either that phorbol esters are not able to fully activate these enzymes or that their substrate specificities are very different from those of the group A enzymes. PKC-zeta, a polypeptide of 80 kDa, does not bind phorbol ester and does not phosphorylate these substrates to a significant extent. Consistent with their ability to bind phorbol ester, recombinant PKC-delta and PKC-epsilon are down-regulated in COS cells by prolonged treatment with phorbol ester, whereas PKC-zeta protein levels remain unaltered.