Anne-Marie Hutchins

Candidate tumor-suppressor genes on chromosome arm 8p in early-onset and high-grade breast cancers

Loss of genetic material from chromosome arm 8p occurs commonly in breast carcinomas, suggesting that this region is the site of one or more tumor-suppressor genes (TSGs). Comparative genomic hybridization analysis showed that 8p loss is more common in breast cancers from pre-menopausal compared with post-menopausal patients, as well as in high-grade breast cancers, regardless of the menopausal status. Subsequent high-resolution gene expression profiling of genes mapped to chromosome arm 8p, on an extended cohort of clinical tumor samples, indicated a similar dichotomy of breast cancer clinicopathologic types. Some of these genes showed differential downregulation in early-onset and later-onset, high-grade cancers compared with lower-grade, later-onset cancers. Three such genes were analysed further by in situ technologies, performed on tissue microarrays representing breast tumor and normal tissue samples. PCM1, which encodes a centrosomal protein, and DUSP4/MKP-2, which encodes a MAP kinase phosphatase, both showed frequent gene and protein loss in carcinomas. In contrast, there was an excess of cases showing loss of expression in the absence of reduced gene copy number of SFRP1, which encodes a dominant-negative receptor for Wnt-family ligands. These candidate TSGs may constitute some of the molecular drivers of chromosome arm 8p loss in breast carcinogenesis.

Genetic aberrations detected by comparative genomic hybridisation in vulvar cancers

Squamous cell carcinoma of the vulva is a disease of significant clinical importance, which arises in the presence or absence of human papillomavirus. We used comparative genomic hybridisation to document non-random chromosomal gains and losses within human papillomavirus positive and negative vulvar cancers. Gain of 3q was significantly more common in human papillomavirus-positive cancers compared to human papillomavirus-negative cancers. The smallest area of gain was 3q22–25, a chromosome region which is frequently gained in other human papillomavirus-related cancers. Chromosome 8q was more commonly gained in human papillomavirus-negative compared to human papillomavirus-positive cancers. 8q21 was the smallest region of gain, which has been identified in other, non-human papillomavirus-related cancers. Chromosome arms 3p and 11q were lost in both categories of vulvar cancer. This study has demonstrated chromosome locations important in the development of vulvar squamous cell carcinoma. Additionally, taken together with previous studies of human papillomavirus-positive cancers of other anogenital sites, the data indicate that one or more oncogenes important in the development and progression of human papillomavirus-induced carcinomas are located on 3q. The different genetic changes seen in human papillomavirus-positive and negative vulvar squamous cell carcinomas support the clinicopathological data indicating that these are different cancer types.

Chromosomal gains and losses in ocular melanoma detected by comparative genomic hybridization in an Australian population-based study

To define the location of potential oncogenes and tumor suppressor genes in ocular melanoma we carried out comparative genomic hybridization (CGH) analysis on a population-based series of 25 formalin-fixed, paraffin-embedded primary tumors comprising 17 choroidal, 2 ciliary body, 4 iris, and 2 conjunctival melanomas. Twelve (48%) of the 25 melanomas showed no chromosomal changes and 13 (52%) had at least one chromosomal gain or loss. The mean number of CGH changes in all tumors was 3.3, with similar mean numbers of chromosomal gains (1.5) and losses (1.8). The highest number of chromosomal changes (i.e., nine) occurred in a conjunctival melanoma and included four changes not observed in tumors at any other ocular site (gains in 22q and 11p and losses in 6p and 17p). The most frequent gains in all primary ocular melanomas were on chromosome arm 8q (69%), 6p (31%) and 8p (23%) and the most frequent losses were on 6q (38%), 10q (23%), and 16q (23%). The most common pairing was gain in 8p and gain in 8q, implying a whole chromosome copy number increase; gains in 8p occurred only in conjunction with gains in 8q. The smallest regions of copy number alteration were mapped to gain of 8q21 and loss of 6q21, 10q21, and 16q22. Sublocalization of these chromosomal changes to single-band resolution should accelerate the identification of genes involved in the genesis of ocular melanoma.

Detailed gene copy number and RNA expression analysis of the 17q12-23 region in primary breast cancers.

Chromosome region 17q12–23 commonly shows an increase in DNA copy number in breast cancers, suggesting that several oncogenes are located at this site. We performed a high‐resolution expression array and comparative genomic hybridization analysis of genes mapped to the entire 17q12–23 region, to identify novel candidate oncogenes. We identified 24 genes that showed significant overexpression in breast cancers with gain of 17q12–23, compared to cancers without gain. These genes included previously identified oncogenes, together with several novel candidate oncogenes. FISH analysis using specific gene probes hybridized to tissue arrays confirmed the underlying amplification of overexpressed genes. This high‐resolution analysis of the 17q12–23 region indicates that several established and novel candidate oncogenes, including a Wnt‐signaling pathway member, are amplified and overexpressed within individual primary breast cancer samples. We were also able to confirm the presence of two apparently separate and reciprocally amplified groups of genes within this region. Investigation of these genes and their functional interactions will facilitate our understanding of breast oncogenesis and optimal management of this disease.

Molecular cloning and sequencing of a novel human P2 nucleotide receptor

A novel human P2 nucleotide receptor has been cloned from a T-cell cDNA library. The predicted amino acid sequence shows characteristics of a G-protein-coupled receptor, and shares 88% homology with a recently characterised rat P2 nucleotide receptor sequence. Distinctive features include an extremely short cytoplasmic tail with only one putative protein kinase C phosphorylation site. Northern blot analysis revealed a 1.9 kb transcript expressed in the placenta.

LCC15-MB cells are MDA-MB-435 : a review of misidentified breast and prostate cell lines

Current stocks of the LCC15-MB cell line, which we originally isolated from a human breast-bone metastasis, were found to be genetically matched to the MDA-MB-435 cell line from the Lombardi Cancer Center (MDA-MB-435-LCC) using comparative genomic hybridisation, DNA microsatellite analysis and chromosomal number. LCC15-MB stocks used for our previously published studies as well as the earliest available LCC15-MB cells also showed identity to MDA-MB-435-LCC cells. The original karyotype reported for LCC15-MB cells was considerably different to that of MDA-MB-435 cells, indicating that the original LCC15-MB cells were lost to contamination by MDA-MB-435-LCC cells. Chromosome number is the simplest test to distinguish original LCC15-MB cells (n ∼ 75) from MDA-MB-435 (n ∼ 52). Collectively, our results prove that LCC15-MB cells currently available are MDA-MB-435 cells and we suggest their re-designation as MDA-MB-435-LCC15 cells. We also review the known misclassification of breast and prostate cancer cell lines to date and have initiated a register maintained at http//www.svi.edu.au/cell_lines_registry.doc.

Molecular cloning and sequencing of the gene encoding a sheep arginine vasopressin type 1a receptor

The gene for the sheep arginine vasopressin type 1a (V1a) receptor subtype was cloned from a genomic library. The deduced amino acid sequence shows characteristics of a G-protein coupled receptor and high sequence identity to human and rat V1a receptor sequences (81% and 73%, respectively). Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a tissue distribution consistent with a type V1a receptor. The genomic DNA (7.1 kb) contains a 1586 bp intron between the putative 6th and 7th transmembrane domains.

EFFECTS OF AN ORALLY ACTIVE VASOPRESSIN V1 RECEPTOR ANTAGONIST

1. This paper reports on the in vitro and in vivo characteristics of a non‐peptide vasopressin V1 receptor antagonist 1‐{1‐[4‐(3‐acetylaminopropoxy)benzoyl]‐4‐piperidyl}‐3,4‐dihydro‐2(1H)‐quinolinone (OPC‐21268).

EFFECTS OF ANTI‐EMETICS ON WATER EXCRETION IN HUMANS

1. The anti‐emetic drug metoclopramide has been shown to stimulate secretion of the antidiuretic hormone arginine vasopressin. Since metoclopramide is used to treat nausea, which is another potent stimulus to vasopressin secretion, the aim of this study was to determine whether metoclopramide might limit free water excretion and so cause hyponatraemia.

V1-like [Arg8]vasopressin binding sites occur in rat hepatocyte nuclei

Arginine vasopressin binding site characterisation was performed on purified nuclei and plasma membranes from livers of Sprague-Dawley rats. [125I][d(CH2)5,Sarc7,Arg8]vasopressin, a selective V1 vasopressin receptor antagonist radioligand, bound to the nuclei in a protein concentration and time dependent manner. Scatchard analysis of nuclear binding sites revealed a single binding site with maximal binding site density (Bmax) of 115 +/- 13 fmol/mg protein and affinity (KD) of 5.2 +/- 0.7 nM. Plasma membrane binding demonstrated a Bmax of 529 +/- 25 fmol/mg protein and KD of 1.9 +/- 0.1 nM. The displacement profile for nuclear binding sites using vasopressin analogues was similar to that for plasma membrane binding sites and was typical of a V1 vasopressin receptor type. There was no evidence of V2-like vasopressin receptor binding using [3H]des-Gly-NH9(2)[d(CH2)5,D-Ile2,Ile4,Arg8]vasopressi n, a selective V2 vasopressin receptor radioligand, in the nuclear or membrane fractions. These results suggest the existence of nuclear V1-like vasopressin binding sites.