Cancer-associated alteration of pericentromeric heterochromatin may contribute to chromosome instability.

Many tumors exhibit elevated chromosome mis-segregation termed chromosome instability (CIN), which is likely to be a potent driver of tumor progression and drug resistance. Causes of CIN are poorly understood but probably include prior genome tetraploidization, centrosome amplification and mitotic checkpoint defects. This study identifies epigenetic alteration of the centromere as a potential contributor to the CIN phenotype. The centromere controls chromosome segregation and consists of higher-order repeat (HOR) alpha-satellite DNA packaged into two chromatin domains: the kinetochore, harboring the centromere-specific H3 variant centromere protein A (CENP-A), and the pericentromeric heterochromatin, considered important for cohesion. Perturbation of centromeric chromatin in model systems causes CIN. As cancer cells exhibit widespread chromatin changes, we hypothesized that pericentromeric chromatin structure could also be affected, contributing to CIN. Cytological and chromatin immunoprecipitation and PCR (ChIP-PCR)-based analyses of HT1080 cancer cells showed that only one of the two HORs on chromosomes 5 and 7 incorporate CENP-A, an organization conserved in all normal and cancer-derived cells examined. Contrastingly, the heterochromatin marker H3K9me3 (trimethylation of H3 lysine 9) mapped to all four HORs and ChIP-PCR showed an altered pattern of H3K9me3 in cancer cell lines and breast tumors, consistent with a reduction on the kinetochore-forming HORs. The JMJD2B demethylase is overexpressed in breast tumors with a CIN phenotype, and overexpression of exogenous JMJD2B in cultured breast epithelial cells caused loss of centromere-associated H3K9me3 and increased CIN. These findings suggest that impaired maintenance of pericentromeric heterochromatin may contribute to CIN in cancer and be a novel therapeutic target.

LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development.

In humans, low peak bone mass is a significant risk factor for osteoporosis. We report that LRP5, encoding the low-density lipoprotein receptor-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG). We find that OPPG carriers have reduced bone mass when compared to age- and gender-matched controls. We demonstrate LRP5 expression by osteoblasts in situ and show that LRP5 can transduce Wnt signaling in vitro via the canonical pathway. We further show that a mutant-secreted form of LRP5 can reduce bone thickness in mouse calvarial explant cultures. These data indicate that Wnt-mediated signaling via LRP5 affects bone accrual during growth and is important for the establishment of peak bone mass.

Differentiation-dependent expression of connective tissue growth factor and lysyl oxidase messenger ribonucleic acids in rat granulosa cells.

Searching for novel genes involved in tissue remodeling during ovarian folliculogenesis, we carried out differential display RT-PCR (DDRT-PCR) on RNA from gonadotropin-stimulated rat granulosa cells (GC). GC from preantral and early antral follicles in immature rat ovaries were cultured in serum-free medium containing no hormone (control), recombinant human FSH (10 ng/ml), 5alpha-dihydrotestosterone (DHT; 10(-6) M), or FSH plus DHT. Total cellular RNA was extracted from cells at 6, 12, 24, and 48 h of treatment for DDRT-PCR analysis, corresponding to an estimated 60% saturation of the messenger RNA (mRNA) population. Six distinct complementary DNA clones were obtained that reproduced the DDRT-PCR profile on a Northern blot of the corresponding RNA samples. Two of these clones detected transcripts that were strongly down-regulated by FSH. One corresponded to connective tissue growth factor (CTGF), a cysteine-rich secreted protein related to platelet-derived growth factor that is implicated in mitogenesis and angiogenesis, and a second was identical to lysyl oxidase (LO), a key participant in extracellular matrix deposition. In detailed expression studies, Northern analysis revealed a single, approximately 2.5-kb CTGF transcript maximally suppressed within 3 h of exposure to FSH with or without DHT and two LO transcripts ( approximately 3.8 and approximately 5.2 kb) maximally suppressed at 6 h. DHT alone did not affect CTGF mRNA, but strongly enhanced LO mRNA relative to the control value. In vivo, CTGF and LO transcripts were significantly suppressed in GC 48 h after equine CG injection (10 IU, ip) compared with untreated controls and were further reduced 12 h after administration of additional 10 IU hCG to induce luteinization. In situ hybridization confirmed GC in preantral/early antral follicles as principal sites of CTGF and LO mRNA expression. We conclude that expression of CTGF and LO mRNAs is inversely related to GC differentiation. The encoded proteins probably have roles in the regulation of tissue remodeling and extracellular matrix formation during early follicular development.

DAZ family proteins exist throughout male germ cell development and transit from nucleus to cytoplasm at meiosis in humans and mice.

The human DAZ gene family is expressed in germ cells and consists of a cluster of nearly identical DAZ (deleted in azoospermia) genes on the Y chromosome and an autosomal homolog, DAZL (DAZ-like). Only the autosomal gene is found in mice. Y-chromosome deletions that encompass the DAZ genes are a common cause of spermatogenic failure in men, and autosomal homologs of DAZ are essential for testicular germ cell development in mice and Drosophila. Previous studies have reported that mouse DAZL protein is strictly cytoplasmic and that human DAZ protein is restricted to postmeiotic cells. By contrast, we report here that human DAZ and human and mouse DAZL proteins are present in both the nuclei and cytoplasm of fetal gonocytes and in spermatogonial nuclei. The proteins relocate to the cytoplasm during male meiosis. Further observations using human tissues indicate that, unlike DAZ, human DAZL protein persists in spermatids and even spermatozoa. These results, combined with findings in diverse species, suggest that DAZ family proteins function in multiple cellular compartments at multiple points in male germ cell development. They may act during meiosis and much earlier, when spermatogonial stem cell populations are established.

A rapid protocol for the authentication of isolated differential display RT-PCR CDNAs.

The most time-consuming and problematic step in the overall DDRT-PCR technique is the confirmation that the isolated cDNA clone represents a differentially expressed gene. We have previously suggested that the majority of apparent false positives generated by DDRT-PCR do in fact result from the PCR reamplification of cDNA species which co-migrate with the cDNA of interest, and we have outlined a procedure to effectively eliminate these from further study. However, in situations where RNA is limiting, it is still desirable to confirm that a purified cDNA amplicon does, in fact, represent the originally observed differentially expressed gene prior to embarking on expression studies.

A human DAZ transgene confers partial rescue of the mouse Dazl null phenotype.

In a subset of infertile men, a spectrum of spermatogenic defects ranging from a complete absence of germ cells (sertoli cell only) to oligozoospermia is associated with microdeletions of the DAZ (deleted in azoospermia) gene cluster on human distal Yq. DAZ encodes a testis-specific protein with RNA-binding potential recently derived from a single-copy gene DAZL1 (DAZ-like) on chromosome 3. Y chromosomal DAZ homologues are confined to humans and higher primates. It remains unclear which function unique to higher primate spermatogenesis DAZ may serve, and the functional status of the gene recently has been questioned. To assess the extent of functional conservation we have tested the capacity of a human DAZ gene contained in a 225-kb yeast artificial chromosome to complement the sterile phenotype of the Dazl null mouse (Dazl-/-), which is characterized by severe germ-cell depletion and meiotic failure. Although Dazl-/- mice remained infertile when the DAZ transgene was introduced, histological examination revealed a partial and variable rescue of the mutant phenotype, manifest as a pronounced increase in the germ cell population of the seminiferous tubules and survival to the pachytene stage of meiosis. As well as constituting definitive proof of the spermatogenic role of the DAZ gene product, these findings confirm the high degree of functional conservation between the DAZ and DAZL1 genes, suggesting they may constitute a single target for contraceptive intervention and raising the possibility of therapeutic up-regulation of the DAZL1 gene in infertile men.

Identification of an essential gene encoding a class-V unconventional myosin in Drosophila melanogaster.

Class-V myosins are a unique type of myosin motor with roles in intracellular transport. The mouse dilute gene was the first member of this class to be cloned, with mutations resulting in lightening of the coat colour or neurogenic defects leading to early death. Further examples of class-V myosins have been described in yeast, chicken and rat. Here, we report the cloning of the first class-V myosin from Drosophila. We show that expression of this myosin is predominantly in the adult germ line and early embryo and that the transcript is localised in the oocyte during oogenesis. Genetic and in situ hybridisation experiments have determined that this gene is located in the 43C region. We have evidence that it maps to a mutation in this region with an embryonic lethal phenotype.

Evidence against the involvement of multiple radical generating sites in the expression of the vascular cell adhesion molecule-1.

The present study was undertaken to investigate the hypothesis that multiple oxygen radical generating systems contribute to the tumor necrosis factor (TNF) alpha-stimulated transcriptional activation of the vascular cell adhesion molecule (VCAM)-1 in endothelial cells. Experimental evidence has implicated the cytochrome P450 monooxygenase and a phagocyte type NADPH-oxidase as a source of oxygen radicals in these cells. We show here that endothelial cells exhibit cytochrome P450 activity by measuring the O-dealkylation of the exogenous substrate 7-ethoxyresorufin, but components of the phagocyte-type NADPH oxidase could not be demonstrated in endothelial cells. In that latter respect it was surprising that the NADPH oxidase inhibitor apocynin completely prevented the accumulation of VCAM-1 mRNA. However, we found that apocynin also acts as an inhibitor of cytochrome P450 activity in endothelial cells. Therefore the inhibitory effect of apocynin on the induction of VCAM-1 may no longer be used to demonstrate a role for the NADPH oxidase in this process. Furthermore, different cytochrome P450 inhibitors Co2+, metyrapone, SKF525a decreased the endothelial VCAM-1 expression stimulated by TNFalpha. Also under hypoxic conditions the expression of VCAM-1 was reduced. On this basis we assume that the oxygen dependent step in the intracellular signalling cascade underlying the TNFalpha stimulated transcriptional activation of VCAM-1 resides in the activity of a cytochrome P450 dependent monooxygenase. The finding that the phospholipase A2 inhibitor bromophenacylbromide inhibited the expression of VCAM-1 may indicate that arachidonic acid serves as a substrate for the cytochrome P450 monooxygenase reaction, but further research is needed to elucidate the particular cytochrome P450 family member mediating the expression of VCAM-1.

Synergy between suppressor of Hairless and Notch in regulation of Enhancer of split m gamma and m delta expression.

The Notch signaling pathway is known to regulate cell fate decisions in a variety of organisms from worms to humans. Although several components of the pathway have been characterized, the actual mechanism and molecular results of signaling remain elusive. We have examined the role of the Notch signaling pathway in the transcriptional regulation of two Drosophila Enhancer of split [E(spl)] genes, whose gene products have been shown to be downstream players in the pathway. Using a reporter assay system in Drosophila tissue culture cells, we have observed a significant induction of E(spl) m gamma and m delta expression after cotransfection with activated Notch. Characterization of the 5' regulatory regions of these two genes led to the identification of a number of target sites for the Suppressor of Hairless [Su(H)] protein, a transcription factor activated by Notch signaling. We show that Notch-inducible expression of E(spl) m gamma and m delta both in cultured cells and in vivo is dependent on functional Su(H). Although overexpression of Su(H) augments the level of induction of the reporter genes by activated Notch, Su(H) alone is insufficient to produce high levels of transcriptional activation. Despite the synergy observed between activated Notch and Su(H), the former affects neither the nuclear localization nor the DNA binding activity of the latter.

The raspberry locus encodes Drosophila inosine monophosphate dehydrogenase.

Investigation of an enhancer-trap line exhibiting testis-specific beta-galactosidase expression led to the isolation of the Drosophila gene encoding inosine monophosphate dehydrogenase (IMPD), the rate-limiting enzyme in guanine nucleotide synthesis, which has been implicated in cell cycle control and malignant transformation. Northern and in situ hybridization analysis demonstrated that the gene has a complex expression pattern involving several independently regulated transcripts. Two ubiquitous, but highly ovary enriched, transcripts of 2.5 and 1.9 kb are expressed in the nurse cells and delivered to the oocyte, whilst a 0.9 kb transcript is found exclusively in the testis. The 2.5 kb transcript encodes a 58 kDa protein, which is highly similar in length and sequence to mouse and human IMPDs and is presumably required for GTP synthesis during early embryogenesis. Over-expression of this cDNA in Escherichia coli yielded a product of the predicted size, which was demonstrated to possess IMPD activity in a spectrophotometric assay. The coding capacity of the other transcripts is currently uncertain. We present evidence that IMPD is the product of the raspberry (ras) locus at 9E and the functions of the gene are discussed in relation to the phenotypes of ras mutants.

Angiotensin-converting enzyme activity is increased in lungs of rats with pulmonary hypoplasia and congenital diaphragmatic hernia.

Lung hypoplasia (LH) and pulmonary hypertension are responsible for the high mortality rate in congenital diaphragmatic hernia (CDH) patients. Angiotensin-converting enzyme (ACE) plays a role in the regulation of pulmonary vascular resistance in the postnatal period and might be involved in the development of pulmonary hypertension of the newborn. A study was made of the development of ACE activity spectrophotometrically in a rat model of LH and CDH. It was previously shown that the lungs in this model are hypoplastic and the muscularization of the pulmonary vascular bed is increased. CDH was induced in fetal rats by oral administration of 115 mg/kg Nitrofen to the mother on day 10.5 of pregnancy. Fetuses were delivered by hysterotomy on days 19, 20, 21, and 22. Nitrofen-exposed rats showed significantly lower lung weights and not statistically significant lower total ACE activities than in controls. ACE activity expressed per milligram lung wet weight and per milligram protein was significantly increased compared to controls. ACE converts angiotensin I to the vasoconstrictor angiotensin II, and it inactivates the vasodilator bradykinin. Increased ACE activity may therefore contribute to pulmonary hypertension. Whether ACE and angiotensin II levels are increased in human newborns with a diaphragmatic defect and whether they contribute to the development of persistent pulmonary hypertension has not been studied up till now.

Sex determination in Drosophila melanogaster.

The understanding of sex determination is a fundamental goal in the study of eukaryotic developmental genetics. The mechanisms governing the generation of sexual dimorphism have been well characterized in Drosophila because of its amenability to both genetic manipulation and the application of the techniques of modern molecular genetics. By using classical genetics to search for sex-transforming mutations and by analysing their phenotypes and how they interact, a picture has emerged involving a cascade of regulatory genes. The primary sex determining signal--the ratio of the number of X chromosomes to the number of sets of autosomes--sets this cascade into motion. Genetic evidence has suggested that the intervening genes in this pathway are active in females but not in males, whereas the final gene has active but opposing roles in the two sexes. This bifunctional locus is responsible for the repression of female differentiation genes in males and male differentiation genes in females. The cloning of the key genes of the regulatory cascade and the study of their transcription patterns have revealed that their different functional states in the two sexes do not result from control at the transcriptional level, as might have been expected. Instead, common primary transcripts are produced in male and female flies; these are then differentially spliced to encode sex-specific gene products. In this paper we focus on the contributions of molecular genetics to the understanding of sex determination. Sufficient background is included for the reader to see how the models of the Drosophila sex determination system were first developed. We then show how the application of new technology has complemented the genetic approach and refined our understanding of the system. Current intensive research in this area should lead within the next few years to definitive knowledge at the molecular level of the cascade of differential splicing of regulatory genes, and how this hierarchy ultimately gives rise to the appropriate sex-specific patterns of structural gene expression that underlie sexual dimorphism.

Secondary chordoma of the mandible.

The first case of primary sacrococcygeal chordoma metastasising to the mandible and presenting with obstructed labour is described. The pathological features of this tumour, its clinical presentation and management are discussed.

Possible involvement of the lipid-peroxidation product 4-hydroxynonenal in the formation of fluorescent chromolipids.

The effects of the lipid-peroxidation product 4-hydroxynonenal on the formation of fluorescent chromolipids from microsomes, mitochondria and phospholipids were studied. Incubation of freshly prepared rat liver microsomes or mitochondria with 4-hydroxynonenal results in a slow formation of a fluorophore with an excitation maximum at 360 nm and an emission maximum at 430 nm. The rate and extent of the development of the 430 nm fluorescence can be significantly enhanced by ADP-iron (Fe3+). With microsomes, yet not with mitochondria. NADPH has a catalytic effect similar to that of ADP-iron. Fluorescent chromolipids with maximum excitation and emission at 360/430 nm are also formed during the NADPH-linked ADP-iron-stimulated lipid peroxidation. Phosphatidylethanolamine and phosphatidylserine react with 4-hydroxynonenal revealing a fluorophore with the same spectral characteristics as that obtained in the microsomal and mitochondrial system. The findings suggest that the fluorescent chromolipids formed by lipid peroxidation are not derived from malonaldehyde, but are formed from 4-hydroxynonenal or similar reactive aldehydes via a NADPH and/or ADP-iron-catalysed reaction with phosphatidylethanolamine and phosphatidylserine contained in the membrane.

Ferritin, a physiological iron donor for microsomal lipid peroxidation.

In the process of lipid peroxidation of microsomes induced either by oxygen radicals generated by xanthine oxidase or by NADPH, ferritin is able to donate the necessary iron. The amount of ferritin necessary to catalyze the process of lipid peroxidation is in the physiological range. In contrast to the finding with phospholipid liposomes, catalase hardly stimulates the lipid peroxidation of microsomes.

Comparison of the inactivation of microsomal glucose-6-phosphatase by in situ lipid peroxidation-derived 4-hydroxynonenal and exogenous 4-hydroxynonenal.

1) The effect of 4-hydroxynonenal and lipid peroxidation on the activities of glucose-6-phosphatase and palmitoyl CoA hydrolase were studied. 2) 4-Hydroxynonenal inactivates glucose-6-phosphatase but has no effect on palmitoyl-CoA hydrolase. These effects are similar with those observed during lipid peroxidation of microsomes. 3) The inhibition of glucose-6-phosphatase by 4-hydroxynonenal can be prevented by glutathione but not by vitamin E. The inactivation of glucose-6-phosphatase during lipid peroxidation is prevented by glutathione and delayed by vitamin E. 4) The formation of 4-hydroxynonenal during lipid peroxidation was followed in relation to the inactivation of glucose-6-phosphatase. At 50% inactivation of glucose-6-phosphatase the 4-hydroxynonenal concentration was 1.5 microM. To obtain 50% inactivation of glucose-6-phosphatase by added 4-hydroxynonenal a concentration of 150 microM or 300 microM was needed with a preincubation time of 30 and 60 min, respectively. 5) It is concluded that the glucose-6-phosphatase inactivation during lipid peroxidation can be due to the formation of 4-hydroxynonenal. The formed 4-hydroxynonenal which inactivates glucose-6-phosphatase is located in the membrane. If this mechanism is valid it implies that a functional SH group of glucose-6-phosphatase is layered in the membrane. However, an inactivation of glucose-6-phosphatase by desintegration of the membrane by lipid peroxidation cannot be ruled out.

Studies on cumene hydroperoxide-induced lipid peroxidation in the isolated perfused rat heart.

In the isolated, perfused rat heart, lipid peroxidation, induced by cumene hydroperoxide (Cum OOH), is accompanied by the release of malondialdehyde (MDA). Using a modified perfusion technique resulting in the separate collection of coronary and interstitial effluent, it can be shown that upon Cum OOH (0.5 mM) perfusion there is an immediate release of MDA in the coronary effluent and a delayed release in the interstitial fluid, indicating the susceptibility and coronary vascular tissue towards free radical-induced lipid peroxidation. Perfusion with Cum OOH leads to an initial increase of the coronary flow and a depressed contractility followed by a cardiac arrest concomitantly with the onset of MDA release in the interstitial fluid. Finally, during prolonged perfusion the coronary flow diminishes and contracture of the heart muscle ('stone heart') develops. These phenomena resemble those occurring during the 'calcium paradox'. Although the contractility diminishes immediately after the perfusion with Cum OOH the tissue ATP level and energy charge (formula; see text) remain constant. From the moment of cardiac arrest the ATP and creatine phosphate levels gradually decrease and the energy charge drops simultaneously with the appearance of MDA in the interstitial fluid. In contrast to the calcium paradox there is no simultaneous increase in the myocardial AMP level. Various mitochondrial enzymes (cytochrome c oxidase, monoamine oxidase, carnitinepalmitoyltransferase I and palmitoyl CoA synthetase) were tested and not affected by Cum OOH perfusion. During the development of contracture after 20 min of Cum OOH perfusion massive contraction band necrosis of cardiac tissue occurs. However, overall protein release is lower when compared with the protein release during the calcium paradox.(ABSTRACT TRUNCATED AT 250 WORDS)

The involvement of iron and lipid peroxidation in the pathogenesis of HCB induced porphyria.

Hexachlorobenzene (HCB) induces a porphyria characterized by a diminished activity of the enzyme uroporphyrinogen decarboxylase (URO-D), presumably due to inactivation by reactive metabolites of HCB. We studied the effect of iron on HCB porphyria in female rats, to determine whether the iron dependent process of lipid peroxidation was involved in the pathogenesis of porphyria. We showed that malondialdehyde formation is increased in rat liver tissue of porphyric rats and that high molecular weight proteins due to cross-linking are formed. We also showed that the induction of porphyria by HCB is dependent on the presence of iron. Our findings suggest that lipid peroxidation is involved in the toxicity of HCB and that the aggravating effects of iron on HCB are mediated by lipid peroxidation.

Biochemical genetics of the Lapland dog model of glycogen storage disease type II (acid alpha-glucosidase deficiency).

A recently described canine model (Lapland dog) of glycogen storage disease type II (GSD II, Pompe disease, acid alpha-glucosidase deficiency) was identified with several biochemical genetic methods. Complementation studies in which fibroblasts from a GSD II dog were fused with fibroblasts derived from control dogs and from human patients with different clinical forms of the disease did not lead to restoration of acid alpha-glucosidase activity in the heterokaryon cell populations. These results indicate that acid alpha-glucosidase deficiency is the primary defect in canine GSD II and that there is a close genetic parallelism with human GSD II. Immunotitration analysis of the residual acid alpha-glucosidase activity in the canine GSD II fibroblasts and liver demonstrated that this residual activity was not due to acid alpha-glucosidase enzyme, in which respect canine GSD II was similar to the infantile form of the human disease. Double immunodiffusion studies showed the presence of catalytically inactive acid alpha-glucosidase enzyme protein in canine GSD II. This is consistent with a structural gene mutation. It is concluded that canine GSD II in the Lapland dog is a homologous model of the infantile form of human GSD II, a conclusion in concordance with clinical and pathological studies.