The influence of extracellular matrix and prolactin on global gene expression profiles of primary bovine mammary epithelial cells in vitro.

An in vitro bovine mammosphere model was characterized for use in lactational biology studies using a functional genomics approach. Primary bovine mammary epithelial cells cultured on a basement membrane, Matrigel, formed three-dimensional alveoli-like structures or mammospheres. Gene expression profiling during mammosphere formation by high-density microarray analysis indicated that mammospheres underwent similar molecular and cellular processes to developing alveoli in the mammary gland. Gene expression profiles indicated that genes involved in milk protein and fat biosynthesis were expressed, however, lactose biosynthesis may have been compromised. Investigation of factors influencing mammosphere formation revealed that extracellular matrix (ECM) was responsible for the initiation of this process and that prolactin (Prl) was necessary for high levels of milk protein expression. CSN3 (encoding kappa-casein) was the most highly expressed casein gene, followed by CSN1S1 (encoding alphaS1-casein) and CSN2 (encoding beta-casein). Eighteen Prl-responsive genes were identified, including CSN1S1, SOCS2 and CSN2, however, expression of CSN3 was not significantly increased by Prl and CSN1S2 was not expressed at detectable levels in mammospheres. A number of novel Prl responsive genes were identified, including ECM components and genes involved in differentiation and apoptosis. This mammosphere model is a useful model system for functional genomics studies of certain aspects of dairy cattle lactation.

Loss of mammary epithelial prolactin receptor delays tumor formation by reducing cell proliferation in low-grade preinvasive lesions.

Top quartile serum prolactin levels confer a twofold increase in the relative risk of developing breast cancer. Prolactin exerts this effect at an ill defined point in the carcinogenic process, via mechanisms involving direct action via prolactin receptors within mammary epithelium and/or indirect action through regulation of other hormones such as estrogen and progesterone. We have addressed these questions by examining mammary carcinogenesis in transplants of mouse mammary epithelium expressing the SV40T oncogene, with or without the prolactin receptor, using host animals with a normal endocrine system. In prolactin receptor knockout transplants the area of neoplasia was significantly smaller (7 versus 17%; P < 0.001 at 22 weeks and 7 versus 14%; P = 0.009 at 32 weeks). Low-grade neoplastic lesions displayed reduced BrdU incorporation rate (11.3 versus 17% P = 0.003) but no change in apoptosis rate. Tumor latency increased (289 days versus 236 days, P < 0.001). Tumor frequency, growth rate, morphology, cell proliferation and apoptosis were not altered. Thus, prolactin acts directly on the mammary epithelial cells to increase cell proliferation in preinvasive lesions, resulting in more neoplasia and acceleration of the transition to invasive carcinoma. Targeting of mammary prolactin signaling thus provides a strategy to prevent the early progression of neoplasia to invasive carcinoma.

A distinct molecular profile associated with mucinous epithelial ovarian cancer.

Mucinous epithelial ovarian cancers (MOC) are clinically and morphologically distinct from the other histological subtypes of ovarian cancer. To determine the genetic basis of MOC and to identify potential tumour markers, gene expression profiling of 49 primary ovarian cancers of different histological subtypes was performed using a customised oligonucleotide microarray containing >59 000 probesets. The results show that MOC express a genetic profile that both differs and overlaps with other subtypes of epithelial ovarian cancer. Concordant with its histological phenotype, MOC express genes characteristic of mucinous carcinomas of varying epithelial origin, including intestinal carcinomas. Differences in gene expression between MOC and other histological subtypes of ovarian cancer were confirmed by RT-PCR and/or immunohistochemistry. In particular, galectin 4 (LGALS4) was highly and specifically expressed in MOC, but expressed at lower levels in benign mucinous cysts and borderline (atypical proliferative) tumours, supporting a malignant progression model of MOC. Hence LGALS4 may have application as an early and differential diagnostic marker of MOC.

A comparison of microarray databases.

Microarray technology has become one of the most important functional genomics technologies. A proliferation of microarray databases has resulted. It can be difficult for researchers exploring this technology to know which bioinformatics systems best meet their requirements. In order to obtain a better understanding of the available systems, a survey and comparative analysis of microarray databases was undertaken. The survey included databases that are currently available, as well as databases that should become available in early 2001. Databases fall into three categories: (i) those that can be installed locally, (ii) those available for public data submission and (iii) those available for public query. Developers of microarray gene-expression databases were asked questions regarding the scope and availability of their database, its system requirements, its future compliance with MGED (Microarray Gene Expression Database) standards, and its associated analytical tools. Participants included AMAD (Stanford/Berkeley/UCSF), ArrayExpress (EBI), ChipDB (MIT/Whitehead), GeneX (NCGR), GeNet (Silicon Genetics), GeneDirector (BioDiscovery), GEO (NCBI), GXD (Jackson Laboratory), mAdb (NCI), maxdSQL (University of Manchester), NOMAD (UCSF), RAD (University of Pennsylvania) and SMD (Stanford University). Other database developers were contacted but data was not available at the time of manuscript preparation. Each database fulfils a different role, reflecting the widely varying needs of microarray users.

Histone- and protamine-DNA association: conservation of different patterns within the beta-globin domain in human sperm.

Most DNA in human sperm is bound to highly basic proteins called protamines, but a small proportion is complexed with histones similar to those found in active chromatin. This raises the intriguing possibility that histones in sperm are marking sets of genes that will be preferentially activated during early development. We have examined the chromatin structure of members of the beta-globin gene family, which are expressed at different times in development, and the protamine 2 gene, which is expressed in spermatids prior to the widespread displacement of histones by transition proteins. The genes coding for epsilon and gamma globin, which are active in the embryonic yolk sac, contain regions which are histone associated in the sperm. No histone-associated regions are present at the sites tested within the beta- and delta-globin genes which are silent in the embryonic yolk sac. The trends of histone or protamine association are consistent for samples from the same person, and no significant between-subject variations in these trends are found for 13 of the 15 fragments analyzed in the two donors. The results suggest that sperm chromatin structures are generally similar in different men but that the length of the histone-associated regions can vary. The association of sperm DNA with histones or protamines sometimes changes within as little as 400 bp of DNA, suggesting that there is fine control over the retention of histones.

X-chromosome activity: impact of imprinting and chromatin structure.

The analysis of the imprinting of the X chromosome has provided insight into factors that affect the initiation and the choice of the chromosome for inactivation in the early mammalian embryo (Lyon, 1996). There are significant differences in the chromatin configuration, methylation and gene expression between Xi and Xa in somatic cells. Preferential paternal X inactivation that is concomitant with widespread heterochromatinization first occurs in the trophectoderm in the blastocyst. It is now clear that the activity of some paternal X-linked genes are suppressed before this stage. In the epiblast there may be early preferential paternal X inactivation before a random pattern supersedes. These observations suggest that parent-specific modification of the chromosome may determine the choice of which X chromosome is to be inactivated (Lyon, 1996). Differential methylation within the Xist gene or the XIC may lead to imprinted X-chromosome behavior. Alternatively, we postulate that imprinting of the X chromosome may be related to differences in chromatin configuration of the X chromosome in male and female germ cells which may then influence X-linked gene expression in the early embryo (Fig. 4). This may occur with a gene by gene effect leading to suppression of paternal alleles. An overall chromatin difference in the chromosomes may influence imprinted paternal Xist expression in early embryos and in the trophectoderm and primary endoderm populations that segregate early from the totipotent progenitors. Alternatively more specific differences in the chromatin architecture of the Xist gene or other gene loci in the Xic may constitute the signature of the imprint.

Perioperative complications in patients with sickle cell disease. An orthopedic perspective.

Osteonecrosis of the femoral head has been reported to occur in 19% to 31% of patients with sickle cell disease, with the condition often being bilateral. Current surgical options for these patients include various forms of arthrodesis, resection arthroplasty, osteotomy, and uncemented hip arthroplasty. Poor surgical outcome, coupled with frequent perioperative medical complications, makes the treatment of these patients very challenging. A case report of a 33-year-old black woman with a 21-year history of sickle cell disease who underwent hip arthroplasty and developed sickle chest syndrome is presented.

Transcripts and CpG islands associated with the pro-opiomelanocortin gene and other neurally expressed genes.

DNA sequences of vertebrate genes which code for neural or neuroendocrine peptides were analysed in terms of CpG dinucleotide distribution and G+C content. The vast majority of the genes were found to contain a region with the sequence characteristics of a CpG island surrounding the 5' end. In mammalian species, the gene which codes for the neuroendocrine polypeptide pro-opiomelanocortin (POMC) was shown to be associated with two separate CpG islands: a 5' CpG island which surrounds the POMC transcription start site and a 3' CpG island which lies approximately 5 kb downstream, encompassing the third exon of POMC. Short POMC-related transcripts, known to be transcribed in the germline, were found to initiate from a promoter within the 3' CpG island. The start sites of the short POMC-related transcripts in mouse testis were mapped to the region coding for gamma MSH in exon 3, in a similar location to transcription start sites identified in other mammalian POMC genes. Similar short POMC-related transcripts were identified in both the mouse F9 embryonal carcinoma cell line and mouse embryonic stem cells, suggesting that transcription initiating within the third exon may occur very early in development. No short transcripts were detected by Northern blot hybridization in either Xenopus laevis testis or oocyte poly(A)+ RNA extracts. The Xenopus laevis POMC genes, A and B, were associated with neither a 5' nor a 3' CpG island. Hence, the presence of a 5' CpG island is not required for production of full-length transcripts from the Xenopus laevis POMC gene, but the presence of a 3' CpG island may be required for transcription to occur from the third exon.

CpG islands in vertebrate genomes.

Although vertebrate DNA is generally depleted in the dinucleotide CpG, it has recently been shown that some vertebrate genes contain CpG islands, regions of DNA with a high G+C content and a high frequency of CpG dinucleotides relative to the bulk genome. In this study, a large number of sequences of vertebrate genes were screened for the presence of CpG islands. Each CpG island was then analysed in terms of length, nucleotide composition, frequency of CpG dinucleotides, and location relative to the transcription unit of the associated gene. CpG islands were associated with the 5' ends of all housekeeping genes and many tissue-specific genes, and with the 3' ends of some tissue-specific genes. A few genes contained both 5' and 3' CpG islands, separated by several thousand base-pairs of CpG-depleted DNA. The 5' CpG islands extended through 5'-flanking DNA, exons and introns, whereas most of the 3' CpG islands appeared to be associated with exons. CpG islands were generally found in the same position relative to the transcription unit of equivalent genes in different species, with some notable exceptions. The locations of G/C boxes, composed of the sequence GGGCGG or its reverse complement CCGCCC, were investigated relative to the location of CpG islands. G/C boxes were found to be rare in CpG-depleted DNA and plentiful in CpG islands, where they occurred in 3' CpG islands, as well as in 5' CpG islands associated with tissue-specific and housekeeping genes. G/C boxes were located both upstream and downstream from the transcription start site of genes with 5' CpG islands. Thus, G/C boxes appeared to be a feature of CpG islands in general, rather than a feature of the promoter region of housekeeping genes. Two theories for the maintenance of a high frequency of CpG dinucleotides in CpG islands were tested: that CpG islands in methylated genomes are maintained, despite a tendency for 5mCpG to mutate by deamination to TpG+CpA, by the structural stability of a high G+C content alone, and that CpG islands associated with exons result from some selective importance of the arginine codon CGX. Neither of these theories could account for the distribution of CpG dinucleotides in the sequences analysed. Possible functions of CpG islands in transcriptional and post-transcriptional regulation of gene expression were discussed, and were related to theories for the maintenance of CpG islands as "methylation-free zones" in germline DNA.