Origin of congenital coronary arterio-ventricular fistulae from anomalous epicardial and myocardial development.

Coronary Artery Fistulae (CAFs) are cardiac congenital anomalies consisting of an abnormal communication of a coronary artery with either a cardiac chamber or another cardiac vessel. In humans, these congenital anomalies can lead to complications such as myocardial hypertrophy, endocarditis, heart dilatation, and failure. Unfortunately, despite their clinical relevance, the aetiology of CAFs remains unknown. In this work, we have used two different species (mouse and avian embryos) to experimentally model CAFs morphogenesis. Both conditional Itga4 (alpha 4 integrin) epicardial deletion in mice and cryocauterisation of chick embryonic hearts disrupted epicardial development and ventricular wall growth, two essential events in coronary embryogenesis. Our results suggest that myocardial discontinuities in the embryonic ventricular wall promote the early contact of the endocardium with epicardial-derived coronary progenitors at the cardiac surface, leading to ventricular endocardial extrusion, precocious differentiation of coronary smooth muscle cells, and the formation of pouch-like aberrant coronary-like structures in direct connection with the ventricular lumen. The structure of these CAF-like anomalies was compared with histopathological data from a human CAF. Our results provide relevant information for the early diagnosis of these congenital anomalies and the molecular mechanisms that regulate their embryogenesis.

Histone H4 lysine 20 monomethylation promotes transcriptional repression by L3MBTL1.

Lethal 3 malignant brain tumor 1 (L3MBTL1), a homolog of the Drosophila polycomb tumor suppressor l(3)mbt, contains three tandem MBT repeats (3xMBT) that are critical for transcriptional repression. We recently reported that the 3xMBT repeats interact with mono- and dimethylated lysines in the amino termini of histones H4 and H1b to promote methylation-dependent chromatin compaction. Using a series of histone peptides, we now show that the recognition of mono- and dimethylated lysines in histones H3, H4 and H1.4 (but not their trimethylated or unmodified counterparts) by 3xMBT occurs in the context of a basic environment, requiring a conserved aspartic acid (D355) in the second MBT repeat. Despite the broad range of in vitro binding, the chromatin association of L3MBTL1 mirrors the progressive accumulation of H4K20 monomethylation during the cell cycle. Furthermore, transcriptional repression by L3MBTL1 is enhanced by the H4K20 monomethyltransferase PR-SET7 (to which it binds) but not SUV420H1 (an H4K20 trimethylase) or G9a (an H3K9 dimethylase) and knockdown of PR-SET7 decreases H4K20me1 levels and the chromatin association of L3MBTL1. Our studies identify the importance of H4K20 monomethylation and of PR-SET7 for L3MBTL1 function.

Frequent gain of chromosome 19 in megakaryoblastic leukemias detected by comparative genomic hybridization.

Acute megakaryocytic leukemia is a rare subtype of AML that is often difficult to diagnose; it is most commonly associated with Down syndrome in children. To identify chromosomal imbalances and rearrangements associated with acute megakaryocytic leukemia, we used G-banding, comparative genomic hybridization (CGH), and whole chromosome painting (WCP) on a variety of primary patients' samples and leukemia cell lines. The most common abnormality was gain of chromosome 19 or arm 19q, which was detected by CGH in four of 12 (33.3%) primary samples and nine of 11 (81.8%) cell lines. In none of the primary samples was this abnormality detected by G-banding analysis. WCP was used to define further the nature of the chromosome 19 gain in the cell lines, which was found to be due to the presence of additional 19q material on marker chromosomes or to cryptic translocations involving 19q. The most common chromosomal loss--detected only in the cell lines--was deletion of chromosomal band 13q14, which was seen in six of 11 (54.5%) cell lines. Other recurrent changes included gains of 1p, 6p, 8q, 11q, 15q, 17q, and 21q and losses of 2, 4q, 5q, 7q, 9p, and 11p. Combining conventional and molecular cytogenetic analyses defined recurrent clonal chromosomal abnormalities, which will aid in the identification of critical genes that are abnormal in acute megakaryocytic leukemia cells.

Identification of candidate genes on chromosome band 20q12 by physical mapping of translocation breakpoints found in myeloid leukemia cell lines.

Deletions of the long arm of chromosome 20 have been reported in a wide range of myeloid disorders and may reflect loss of critical tumor suppressor gene(s). To identify such candidate genes, 65 human myeloid cell line DNAs were screened by polymerase chain reaction (PCR) for evidence of allelic loss at 39 highly polymorphic loci on the long arm of chromosome 20. A mono-allelic pattern was present in eight cell lines at multiple adjacent loci spanning the common deleted regions (CDRs) previously defined in primary hematological samples, suggesting loss of heterozygosity (LOH) at 20q. Fluorescence in situ hybridization (FISH) was then performed using a series of yeast artificial chromosomes (YACs) ordered in the CDR, and in five of eight cell lines, the deletions resulted from cytogenetically detectable whole chromosomal loss or large interstitial deletion, whereas in another cell line deletion was associated with an unbalanced translocation. LOH in the CMK megakaryocytic cell line, which has a hypotetraploid karyotype, was associated with a der(20)t(1;20)(q32;q12)x2 leading to complete deletion of the CDR. Three additional unbalanced translocations were found within the CDR and all three breakpoints mapped to a single YAC. We then used a series of P1 artificial chromosomes (PACs) spanning this YAC clone, and two PACs produced 'split' signals suggesting that they each span one of these breakpoints. Exon trapping using PACs that overlap the breakpoint regions yielded portions of six genes and evaluation of these genes as candidate tumor suppressor genes is underway. The limited information available about these genes suggests that the h-l(3)mbt gene is the most attractive candidate.

Comparative mutational analysis of DPC4 (Smad4) in prostatic and colorectal carcinomas.

Allelic deletions of chromosome 18q are reported to be common in prostate and colorectal cancers, suggesting that one or more tumor suppressor genes on 18q are involved in the genesis of these neoplasms. The DPC4 gene, a recently identified candidate tumor suppressor in 18q21, was examined for evidence of inactivation in prostatic carcinomas, and results compared to those of a parallel analysis of colorectal carcinomas, for which DPC4 mutation has been reported in approximately 10% of cases. In this study, only three (10%) of 29 informative primary prostate cancers showed allelic loss of chromosome 18q21 markers, and no point mutations or deletions of DPC4 were detected in the complete set of 45 primary or metastatic cases. In contrast, five (56%) of nine primary colorectal tumors displayed allelic loss of 18q markers and in one of these a somatically acquired G-->T missense mutation was found in exon 1. Of twelve colorectal tumor cell lines, one showed a G-->C missense mutation in exon 8 and two had partial homozygous deletions that would likely abrogate gene function. These data suggest that DPC4 is rarely if ever mutated during prostatic oncogenesis, whereas inactivation of this gene may contribute to the genesis of a subset of colorectal carcinomas.

Tumour-suppressor genes in prostatic oncogenesis: a positional approach.

Genetic alterations, such as mutation, methylation and aneuploidy, are thought to underlie the multistep genesis and progression of many human cancers. However, the genetic events occurring in prostatic oncogenesis are still relatively poorly understood. This is especially so in early-stage tumours, in which mutations of known oncogenes or tumour-suppressor genes appear to be quite infrequent. Allelic losses of chromosome arms 7q, 8p, 10, 16q and 18q suggest the involvement of novel suppressor loci on these chromosomes; allelic losses of chromosome arm 8p are especially frequent and may be detected even in early-stage tumours. We have used a positional approach to seek novel genetic targets in prostate cancer, including allelic-loss mapping of chromosome 8p and physical mapping of chromosome band 8p22 around the MSR gene. A homozygous somatic deletion in one prostatic nodal metastasis was mapped in this region and spanned 730-970 kb. This region was then examined in detail for expressed sequences. One novel gene, called N33, was found to be silenced by a methylation mechanism in most colon cancer cell lines and some primary colorectal tumours. Characterization of additional chromosome 8p22 candidates is in progress.

Tumour suppressor genes in prostate cancer.

Inactivation of tumour suppressor gene function is a critical step in the development of human neoplasia. The Rb and CDKN2 tumour suppressor genes are inactivated in many tumour types, including the late stages of prostate cancer, and appear to function in the same suppressor pathway. p53, another major tumour suppressor is also mutated in a subset of advanced-stage prostate carcinomas. E-cadherin and other cell adhesion genes, which have been characterized as suppressors of the metastatic phenotype, are inactivated or downregulated during progression to advanced prostate cancer and have been associated with poor clinical outcome. The early genetic events involved a prostatic neoplasia are poorly understood, but loss of as yet undiscovered tumour suppressor genes may play a role in the initiation of this disease.

Physical mapping of chromosome 8p22 markers and their homozygous deletion in a metastatic prostate cancer.

Numerous studies have implicated the short arm of chromosome 8 as the site of one or more tumor suppressor genes inactivated in carcinogenesis of the prostate, colon, lung, and liver. Previously, we identified a homozygous deletion on chromosome 8p22 in a metastatic prostate cancer. To map this homozygous deletion physically, long-range restriction mapping was performed using yeast artificial chromosomes (YACs) spanning approximately 2 Mb of chromosome band 8p22. Subcloned genomic DNA and cDNA probes isolated by hybrid capture from these YACs were mapped in relation to one another, reinforcing map integrity. Mapped single-copy probes from the region were then applied to DNA isolated from a metastatic prostate cancer containing a chromosome 8p22 homozygous deletion and indicated that its deletion spans 730-970 kb. Candidate genes PRLTS (PDGF-receptor beta-like tumor suppressor) and CTSB (cathepsin B) are located outside the region of homozygous deletion. Généthon marker D8S549 is located approximately at the center of this region of homozygous deletion. Two new microsatellite polymorphisms, D8S1991 and D8S1992, also located within the region of homozygous deletion on chromosome 8p22, are described. Physical mapping places cosmid CI8-2644 telomeric to MSR (macrophage scavenger receptor), the reverse of a previously published map, altering the interpretation of published deletion studies. This work should prove helpful in the identification of candidate tumor suppressor genes in this region.

Structure and methylation-associated silencing of a gene within a homozygously deleted region of human chromosome band 8p22.

The structure and expression pattern of a human gene located within a homozygously deleted region of a metastatic prostate cancer have been characterized. Multiple cDNA fragments of this gene were isolated by hybrid capture with yeast artificial chromosome clones covering the deletion region. Eleven coding exons spanned 205-220 kb of the 730- to 970-kb deletion. The predicted amino acid sequence was 43% identical to that of an anonymous Caenorhabditis elegans gene and 20% identical to an accessory or regulatory subunit of the oligosaccharyltransferase enzyme complex in Saccharomyces cerevisiae. Hydrophobicity profiles of all three gene products were similar and showed four putative membrane-spanning domains in the molecules' C-terminal halves, suggesting a general conservation of function. The gene was expressed as an approximately 1.5-kb mRNA in most nonlymphoid human cells/tissues including prostate, lung, liver, and colon. Expression was detected in many epithelial tumor cell lines, but was undetectable by Northern blot or RT-PCR in 14 of 15 colorectal, 1 of 8 lung, and 1 of 4 liver cancer cell lines. Lack of expression in tumor cell lines was highly correlated with hypermethylation of a CpG island located at the gene's 5' end. These findings form a basis for further work on this candidate tumor suppressor gene.

Yeast artificial chromosome and radiation hybrid map of loci in chromosome band 8p22, a common region of allelic loss in multiple human cancers.

Polymorphic alleles at loci such as LPL (lipoprotein lipase) and MSR (macrophage scavenger receptor) in chromosome band 8p22 are frequently lost during the genesis of several types of human cancer, including colorectal, non-small cell lung, hepatocellular, and prostatic carcinomas. A physical map of 31 published or novel probes and sequence-tagged sites in this genetic region was constructed using a radiation hybrid panel and the CEPH (Centre d'Etude du Polymorphisme Humain) yeast artificial chromosome (YAC) library. Thirty-six overlapping YACs defined a physical order for the following polymorphic markers: tel-D8S26-D8S511-D8S549-MSR-D8S254-D8S233- D8S261-D8S21-LPL-D8S258-cen. These maps unify small consensus regions of allelic loss on chromosome 8p defined by restriction fragment length polymorphisms with more informative PCR-based polymorphisms and widely available YAC mapping resources.

Comparative genomic hybridization, allelic imbalance, and fluorescence in situ hybridization on chromosome 8 in prostate cancer.

Due to problems with primary tumor cell culture, conventional cytogenetics has yielded little insightful information on chromosomal alterations in prostate cancer. The primary aim of this study was to define the ability of comparative genomic hybridization (CGH) to detect and map genetic deletions in prostate tumors. A secondary aim was to apply multiple assays to individual tumors as a means of deciphering the mechanisms of genetic alterations in prostate cancer. CGH results were compared with allelic imbalance measurements at 29 distinct loci on chromosome 8 in 18 specimens (17 malignant and 1 benign). CGH detected no changes in cases where all informative PCR/RFLP loci were retained and detected all p arm deletions consisting of at least two loci. We estimate that in this study, the smallest deletions detected by CGH were approximately 20-30 cM. Physical mapping of subchromosomal arm deletions by CGH correlated well with allelic imbalance mapping by PCR/RFLP: The data agreed at 88% of loci on 8p and 92% of loci on 8q. Fluorescence in situ hybridization (FISH) with multiple centromere probes and DNA content flow cytometry (FCM) also was performed on selected specimens. FISH revealed two cases of chromosome 8 aneusomy. In these two cases and three others, CGH showed simultaneous p arm deletion and q arm gain, suggesting isochromosome 8q formation. Together, these data suggested that, simple chromosomal aberrations were responsible for allelic losses on 8p and allelic gains on 8q in a significant number of prostate tumors. We also used CGH to examine relative DNA sequence copy number throughout the genome. Changes frequently associated with 8p loss include gains of 8q and losses of 13q, 16p, 16q, 17p, 17q, 20q, and Y. Cases with 8p loss exhibited five times the number of alterations as did cases without 8p loss.

Loss of chromosome arm 8p loci in prostate cancer: mapping by quantitative allelic imbalance.

A previous study of 18 primary or metastatic prostate cancers showed loss of genetic markers on chromosome 8; 10, or 16 in more than 50% of cases [Bergerheim USR et al. (1991) Genes Chromosom Cancer 3:215-220]. The small size and infiltrative nature of primary prostatic tumors have hindered efforts to assess allelic losses by traditional restriction fragment length polymorphism (RFLP)/Southern blotting methods. To improve the sensitivity and specificity of this analysis in early prostate cancer, we have amplified polymorphic microsatellite repeats by polymerase chain reaction (PCR), and have quantitated allelic imbalances with phosphor imaging technology. In this study, 63 primary prostate tumors and matched benign tissues obtained by radical prostatectomy were examined at 28 genetic loci on chromosome 8, all but five of which were located on the short arm. Twenty-nine (46%) of the 63 cases showed loss of at least one locus. Multiple adjacent loci, usually including the LPL and MSR genes in 8p22, were lost in 28 cases. In 10 of these, losses were observed at all informative loci on the p arm. In another 15 tumors, losses were restricted to subregions of the p arm by loci retained either distally toward the p terminus or proximally at the 8p12-8p21 border, or both. In three tumors, two discrete regions of loss were observed within 8p, separated by several retained loci. Allelic loss of 8p loci was associated with higher tumor grade. These data are complementary to previous reports of allelic deletions in colorectal, hepatocellular, and non-small cell lung cancers and suggest the existence of one or more pleotropic tumor suppressor genes on 8p.

Expression and functionality of the trkA proto-oncogene product/NGF receptor in undifferentiated hematopoietic cells.

The expression of the low-affinity NGF receptor (p75) and the trkA proto-oncogene product was analyzed in a series of human hematopoietic cell lines at protein and RNA levels. We did not detect any form of NGF receptor in cell lines displaying a myelomonocytic phenotype (HL60 and U937). In contrast, cells displaying a more immature erythroleukemic phenotype (TF1 and K562) expressed TrkA in the absence of detectable p75. Scatchard analysis showed a single high-affinity site for NGF (kd = 10(-10) mol/L), with a copy number ranging from 300 to 3,000 sites per cell depending on the studied cell line. In addition, NGF induced autophosphorylation of TrkA and could substitute for granulocyte-monocyte colony-stimulating factor to trigger the proliferation of the TF1 cell line, with a half-maximal signal observed at 50 pmol/L, indicating that p75 is not required for DNA synthesis in this cell line. The physiologic relevance of NGF in early hematopoiesis was confirmed by showing that 12% to 15% of progenitor blood cells from mice treated with 5-fluorouracil expressed TrkA and that these cells could be induced to proliferate and differentiate in response to NGF in association with macrophage colony-stimulating factor. Our study demonstrates for the first time that trkA proto-oncogene expression and activation is not restricted to the nervous system, but is also an important element in early hematopoiesis.

p53 is mutated in a subset of advanced-stage prostate cancers.

Inactivation of p53, a tumor suppressor gene, contributes to the genesis and/or progression of a substantial fraction of all human cancers, including > or = 50% of breast, lung, and colon carcinomas. Mutated p53 alleles typically contain missense single-base substitutions within exons 5-8 and encode abnormally stable p53 proteins that accumulate to high levels in tumor cell nuclei. To evaluate the frequency, type, and clinical significance of p53 mutation in human prostate cancer, archival tumor material from 150 prostate cancer patients was examined by immunohistochemistry (IHC) with anti-p53 antibodies. Abnormal nuclear p53 accumulation (IHC) was observed in 19 tumors (12.7%) and was strongly related to disease stage (23% of 69 stage III or IV tumors were IHC+ versus 4% of 74 stage 0-II tumors; P < 0.001, Fisher's exact test). The methods of polymerase chain reaction, single-strand conformational polymorphism, and direct sequencing were used to identify mutations, predominantly missense single-base substitutions in exons 5, 7, or 8 in 9 of 14 IHC+ cases but in none of 20 IHC- cases; 5 of these mutations were G:C-->A:T transitions at CpG dinucleotides. These data indicate that mutated p53 alleles are quite uncommon in early prostate cancers but are found in 20-25% of advanced cancers, suggesting a role for p53 mutation in the progression of at least a subset of prostate cancers.

Expression of nerve growth factor and nerve growth factor receptor genes in human tissues and in prostatic adenocarcinoma cell lines.

Nerve growth factor (NGF) mRNAs were detected and quantified in a variety of normal and neoplastic human tissues by northern blot hybridization. Human heart contained the highest NGF mRNA levels, whereas lower but comparable levels were found in the placenta, prostate, and kidney. All tissues examined coexpressed the low-affinity NGF receptor (LNGFR), whereas none of these tissues expressed the high-affinity NGF receptor encoded by the trk protooncogene. The widespread distribution of the LNGFR suggests that it plays a role in the regulation of normal cell growth. No overexpression of NGF or LNGFR mRNA was detected in neoplastic tissues, whereas LNGFR-like immunoreactivity was localized outside of tumor cells. Transforming growth factor-alpha and protooncogene c-fos expression in these tissues did not show a systematic correlation with NGF/LNGFR expression. Furthermore, regulation of the human NGF gene was studied in DU145 cells, a prostatic adenocarcinoma cell line that synthesizes significant NGF mRNA levels. Serum induced, whereas dexamethasone inhibited, NGF mRNA synthesis in these cells. Serum induction was preceded by a rapid and transient activation of the c-fos protooncogene.

Expression of the beta-nerve growth factor gene in male sex organs of the mouse, rat, and guinea pig.

Steady-state nerve growth factor (NGF) mRNA levels were estimated in male sex organs of the mouse, rat, and guinea pig by RNA blot hybridization analysis. The abundance of NGF mRNAs was in the order vas deferens greater than epididymis greater than or equal to seminal vesicles much greater than testis. NGF mRNA levels in these organs were compared with those estimated for other rat peripheral tissues and were found to correlate with the density of their sympathetic innervation, with the exception of guinea pig prostate. Castration had no significant effect on NGF mRNA levels in the guinea pig prostate, suggesting that NGF synthesis in this tissue is not under direct androgen control. NGF-like and proNGF-like immunoreactivities were localized by immunohistochemical techniques in the secretory cells of the glandular epithelium of the guinea pig prostate and in germ cells in the seminiferous tubules of the mouse testis.

1,25-Dihydroxyvitamin D3 is a potent inducer of nerve growth factor synthesis.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), a metabolically active form of vitamin D, is shown to increase in a dose-dependent manner the cellular pool of NGF mRNA in murine L-929 fibroblasts cultured in a serum-free medium. This effect can be detected as early as 3 hours after 1,25-(OH)2D3 addition and persists for at least 28 hours. It is accompanied by an enhancement of the amount of NGF protein secreted in the culture medium. Since the proto-oncogene c-fos appears involved in the regulation of the NGF gene (Mocchetti et al.: Proceedings of the National Academy of Sciences of the United States of America 86: 3871-895, 1989; Hengerer et al: Proceedings of the National Academy of Sciences of the United States of America 87:3899-3903, 1990), the effect of 1,25-(OH)2D3 on c-fos expression was analysed and compared to that elicited by other inducers of the NGF gene, serum (Wion et al: FEBS Letters 189:37-41, 1985) and phorbol 12-myristate 13-acetate (PMA) (Wion et al: FEBS Letters 262:42-44, 1990). Addition of serum or PMA to L-929 cells was rapidly followed by a transient activation of the c-fos gene. In contrast, c-fos transcripts remained undetected in the presence of 1,25-(OH)2D3. The failure to find any evidence of c-fos expression suggests that 1,25-(OH)2D3 could enhance the pool of NGF mRNA by a mechanism independent of the c-fos pathway.(ABSTRACT TRUNCATED AT 250 WORDS)