Modification of gene expression induced by siRNA targeting of estrogen receptor alpha in MCF7 human breast cancer cells.

To establish a model of endocrine resistant breast cancer that is associated with loss of estrogen receptor (ER), MCF7 cells were transfected with several plasmid constructs intended to produce intracellular double stranded hairpin RNA to be processed into siRNA directed against different regions of the ERalpha mRNA. Stably transformed cells were propagated in long-term culture. One of these lines, designated pII, was selected for further analysis. pII cells exhibited reduced levels of ERalpha mRNA and protein as well as several estrogen-regulated genes assessed by real-time PCR and were unresponsive to addition of estradiol and tamoxifen. Higher levels of ERbeta were measurable as compared with parental MCF7 cells. There was an unexpected decrease in expression in members of the EGFR family in contrast with observations reported for ER-negative tumours or some other established endocrine-independent lines. Microarray gene analysis comparing expression in parental MCF7 with pII cells in both serum-synchronised and non-synchronised conditions highlighted a spectrum of other genes that were expressed at different levels compared to the parental MCF7 cells. Genes showing the greatest change were mostly common between synchronized and unsynchronised cells; GRB7, PSMD7, KRT19, KRT18, AKT1, SYNCRIP, CYB5A and EVL for down-regulated in pII and QDPR, VIM, CD68, CA9, STMN1, CDK2, CTSC for up-regulated in pII cells. Notably, the decreased expression of epithelial keratins 18 and 19 and an increase in vimentin and in a macrophage marker CD68, is suggestive of an epithelial to mesothelial transition. Further characterisation of these cells particularly with respect to the factors controlling their growth may contribute to a better understanding of the behaviour of cells that have become endocrine independent by loss of ER function.

Expression of growth factor and epidermal growth factor receptor encoded transcripts in human gastric tissues.

Expression of mRNA-encoding transforming growth factors alpha and beta (TGF alpha and beta), epidermal growth factor receptor (EGFR), and platelet-derived growth factors (PDGF) A and B chains was examined in 63 human gastric biopsies. Despite considerable individual variation, transcript levels were generally higher in 16 paired gastric tumors compared with surrounding epithelium. Marked increases were observed for the TGFs and c-sis, whereas EGFR mRNA was poorly expressed; there was no correlation with pathological staging of the cancers. In the nonneoplastic tissues, 14 had normal histology and 27 displayed superficial (SG) or atrophic gastritis (AG). Transcript levels greater than or equal to + were similar between these categories for all the growth factors, but were about 50% higher for EGFR in the tissues with gastritis. Concurrent expression of TGF alpha and EGFR (greater than or equal to + level) was more frequent in the paired tumors (38%) than in adjacent nonmalignant tissue (6%) and was seen in only one of 14 (7%) normal samples, in three of 19 (16%) of those with AG, and none of eight of those displaying SG. High levels of TGF beta and PDGFA mRNA were expressed in gastric ulcers, with little or no TGF alpha and EGFR transcripts; in contrast both TGFs and EGFR message were found in normal oesophagus. Stomach tissues are thus capable of synthesizing a variety of growth factors. These may be associated with nonneoplastic hyperplasia and/or malignant proliferation. Coexpression of TGF alpha/EGFR supports the possibility of an autocrine loop sustaining tumor growth which is different from the mechanisms responsible for normal cellular proliferation.

Interaction of vasopressin and oxytocin with human breast carcinoma cells.

The arginine vasopressin and oxytocin content of normal and cancerous human breast tissue were measured using radioimmunoassay. Both peptides were present in amounts greater than that found in the circulation, but no difference between normal and malignant tissues was found. Binding of [3H]oxytocin and [3H]vasopressin were characterized in human breast carcinoma cells (MCF7 cells). Binding of both hormones to MCF7 cells was specific and saturable, the vasopressin receptor found to be of the V1 subtype. Scatchard analyses of the data were linear, indicating a single high affinity, low capacity binding site for each hormone (vasopressin: KD = 47.4 +/- 1.6 nmol/liter, Bmax = 27,300 +/- 6,500 sites/cell; oxytocin: KD = 51.3 +/- 0.4 nmol/liter, Bmax = 87,000 +/- 4,000 sites/cell). The effects of vasopressin and oxytocin on the growth of MCF7 cells were assessed using protein accumulation and cell numbers. Vasopressin at 10-1000 pmol/liter was mitogenic for MCF7 cells, but higher doses (10 nmol/liter) were growth inhibitory. Oxytocin was also mitogenic for MCF7 cells but to a lesser extent than vasopressin. In conclusion, we suggest that vasopressin and possibly oxytocin may be important modulators of the growth of some human breast carcinomas.

Expression of FGFR2 BEK and K-SAM mRNA variants in normal and malignant human breast.

The expression of mRNA encoding alternative forms of fibroblast growth factor receptor 2 (FGFR2) differing in the carboxy terminal half of their third immunoglobulin-like domain, was investigated in 77 human breast cancer tissues, 12 non-malignant breast biopsies and 29 cell lines, using a reverse transcriptase (RT) polymerase chain reaction (PCR) method. RNA from the two tissue groups yielded PCR product corresponding to both the BEK and the K-SAM form; amounts normalised to glyceraldehyde phosphate dehydrogenase product were similar in both groups. The level of either variant or of the total FGFR2 product was essentially unrelated to prognosis or clinical status except that patients with advanced clinical T staging had a higher proportion of BEK to K-SAM (P = 0.01). RNA from 1/2 normal breast derived and 8/10 breast cancer cell lines expressed exclusively or predominantly the K-SAM form; 2/10 had significant amounts of both BEK and K-SAM mRNA. Of 12 other epithelial lines, seven expressed mainly K-SAM mRNA, four expressed BEK and one was negative. Of five non-epithelial lines, one was negative, two expressed only BEK mRNA and two had significant amounts of both variants. We conclude that tissue levels of FGFR2 mRNA are unaltered in breast cancer extracts and that the splicing mechanism for this exon selection appears not to be significantly disrupted.

Allelic variation of BAT-25 and BAT-26 mononucleotide repeat loci in tumours from a group of young women with breast cancer.

Genomic instability in the form of repeat number variation at microsatellite loci has been reported in several human cancers. To resolve current confusion regarding frequency of microsatellite instability (MSI), standardised protocols have proposed use of a consensus set of informative loci; it is claimed that analysis of 2 apparently quasi-homozygous, quasi-monomorphic, mononucleotide repeats (BAT-25 and BAT-26) is sufficiently accurate to define MSI (obviating need for corresponding constitutive DNA). We examined these loci in 163 breast cancers, 108 of which had previously been analysed at 11-24 other loci and found to have MSI in 38% of them. For BAT-26 only 1/153 (homozygous) tumours showed a contracted allele, with minor size variations (1-6 bp) between individuals. For BAT-25 repeat contractions were unambiguously observed in 12 (7.4%) tumours; only 4 of these were previously designated MSI+. DNA from normal individuals showed significant allelic variation in 8/159 (5%) cases for BAT-25; no instance of heterozygosity was seen for BAT-26. Subsequently, we analysed normal DNA from the 12 individuals whose tumours had MSI at BAT-25; in 2 cases there was germline heterozygosity. We conclude that analysis with BAT-26 (in contrast to other loci) was not a useful detector of MSI. With BAT-25, a low frequency of MSI not much greater than germline polymorphism, also limits the utility of this marker for determining MSI in breast cancer.

Detection of breast cancer-associated estrone sulfatase in breast cancer biopsies and cell lines using polymerase chain reaction.

Steroid sulfatase (STS) is a single enzyme with a range of substrate specificities, including estrone sulfate. Using a 2.4 kb cDNA clone, expression of human STS was undetectable by Northern hybridization, but STS RNA was detected in human placenta, human breast cancer samples, and in breast carcinoma cell lines following reverse transcriptase-PCR amplification, using specific primers to yield a product of 472 bp. In preliminary studies, stimulation of MCF-7 cell lines with estradiol (10(-8) M) resulted in an increased level of amplifiable STS RNA, and this upregulation of STS RNA could be abolished by tamoxifen. The estrone sulfatase activity in mammary tumors derived from N-nitrosomethylurea (NMU) treated rats was significantly decreased in animals treated with tamoxifen compared to control animals, regardless of the response of the tumors to the antiestrogen (P < 0.05). Although tamoxifen does not inhibit the estrone sulfatase enzyme in vitro, it may modulate the expression of STS RNA and the enzyme activity in vivo.

Epstein-Barr virus in gastric carcinoma and adjacent normal gastric and duodenal mucosa.

AIM: To look for Epstein-Barr virus (EBV) in a series of gastric cancers of common and rare histological types. METHODS: Formalin sections from 19 cases of gastric carcinomas of different types were studied using in situ hybridisation with fluorescein conjugated EBER oligonucleotides and the avidin-biotin complex immunoperoxidase technique, using the monoclonal antibody Dako-EBV CS 1-4. RESULTS: The only positive tumour was a lymphoepithelioma-like gastric carcinoma. The remaining 18 cases, which included 11 consecutive cases of usual adenocarcinomas, three early gastric cancers, two adenosquamous carcinomas, and a case each of signet ring carcinoma and neuroepithelioma, were all negative. However, scattered EBV positive cells were seen in the normal gastric or duodenal mucosa bordering the tumours in seven out of 11 cases. CONCLUSIONS: Lymphoepithelioma-like gastric carcinoma seems to be the main, if not the only, EBV positive gastric cancer. The presence of EBV positive normal gastric and duodenal cells suggests that these cells may act as a reservoir for the virus in some people--a possibility that deserves wider investigation.

Multifocal squamous cell carcinoma of the oesophagus following radiotherapy for bilateral breast carcinoma.

A 60 year old woman who presented with dysphagia and weight loss was found to have multiple foci of dysplasia and in situ and invasive squamous cell carcinoma scattered along the whole length of the oesophagus, with intervening areas of normal mucosa. The patient had a history of two breast carcinomas 19 and one year previously for which she had repeated radiotherapy. Several members of the patient's close family had histories of malignant disease. All oesophageal lesions and the more recent breast cancer showed positive immunostaining for p53 protein. p53 mutations, some involving different exons, were also detected in these lesions. No p53 immunostaining or mutations were detected in the normal oesophageal mucosa. The findings suggest an independent origin of the multiple dysplastic and neoplastic foci, which might have developed in a background of a field change, possibly related to the previous radiotherapy. The strong family history of malignant diseases raises the possibility that, in addition, genetic factors might have played a role in the development of the oesophageal disease.

CD5 positive breast carcinoma in a patient with untreated chronic lymphocytic leukaemia: molecular studies of chromosome 13q.

A 67 year old woman presented with a right breast lump which proved to be a grade 2 invasive ductal carcinoma with axillary lymph node metastasis. She had a five year history of CD5 positive chronic lymphocytic leukaemia, which never required treatment. Immunoperoxidase stains for CD5, using the monoclonal antibody NCL-CD-54C7, showed that there was extensive infiltration of axillary lymph nodes with CD5 positive B lymphocytes. Strong staining for CD5 was also seen in the carcinoma cells within the breast and lymph node metastases. It has recently been suggested that there is a tumour suppresser locus in chronic lymphocytic leukaemia at 13q12.3 near or at the BRCA2 locus. Deletion of regions on chromosome 13q containing the BRCA2 and RB1 genes has also been reported in sporadic breast cancers. These observations suggest that there may be a link between these two diseases acting through chromosome 13, but amplification of several microsatellite repeat markers failed to show any loss of heterozygosity or repeat instability at either these or several other loci on chromosome 13. Examination of additional such cases is needed to perform a more comprehensive study of the significance of positive CD5 staining of breast carcinoma.

On the specificity of uptake by isolated Torpedo synaptic vesicles.

Synaptic vesicles were isolated from the electric organ of Torpedo marmorata in highly purified form. Their uptake properties were examined using a large number of small organic molecules as substrates. Following incubation at 26 degree C for 1 h, it was found that concentrative accumulation, indicated by a vesicle:medium concentration ratio greater than unity, was achieved by all the choline analogues used and by four biogenic amines, but not by a variety of purine and pyrimidine bases and nucleosides. Amino acids penetrated poorly, as did sugars, and of organic anions, acetate but not citrate or thiocyanate, was almost excluded. Thus Torpedo vesicles are relatively impermeable to compounds which cannot utilize the ACh or ATP carriers, but show a very high rate of amine uptake, which may be linked to a pH gradient.

Evaluation of prognostic factors in male breast cancer.

We conducted an analysis on 41 cases of male breast cancer (median age 54 y; range 25-82 y) in Kuwait. Most (51%) were stage II cancers with 65% arising in the left breast. There were 5 (12%) T1 tumours, 23 (56%) T2 tumours and 13 (32%) T3/T4 tumours. They were mostly (95%) infiltrating ductal carcinomas; 97% were grade 2 or 3. Axillary lymph node involvement was found in 69%. Estimated 5-year survival rates were 67% and 58% for overall and relapse free survival respectively. Favourable prognosis was associated with age below 50y, clinical stage I and II, small tumour size (T1, T2), low tumour grade and absence of nodal involvement or distant metastases; nodal status and grade were independent factors for relapse free survival in multivariate analysis. In 18 cases, an immunohistochemical study showed some degree of tumour antigen reaction for ER in 89% of cases, PR in 61%, pS2 in 44%, CathD in 72%, p53 in 56%, c-erbB-2 in 50%, Ki67 and PCNA in 100% and bcl-2 in 78%. There were significant associations between several of these factors but none influenced survival. Despite the high incidence of staining of ER, our data do not support the concept of an endocrine pathway that could be usefully antagonized with antioestrogens for therapeutic benefit, as in women.

Detection of Epstein-Barr virus in gastrectomy specimens.

Epstein Barr virus (EBV) has been reported to be present in a minority of gastric carcinomas and may be implicated in its pathogenesis. This study was aimed at determining the occurrence of EBV in 43 consecutive gastrectomy specimens with a variety of benign and malignant lesions. In situ hybridisation was used for detection of EBER RNA, the marker for latent EBV infection. Only 1/20 (5%) gastric cancers was EBER positive; a moderately differentiated adenocarcinoma with a heavy lymphocytic infiltration. The interstitial lymphocytic infiltrate was predominantly of B cell type, but the majority of lymphocytes overlying the tumour cells were CD8+ T cells. The other gastric lesions examined, which included 15 peptic ulcers, 6 stromal tumours and 2 lymphomas, were all EBER negative. Using a biotin detection system, scattered EBER positive cells were seen in adjacent normal gastric and/or duodenal mucosa in 9 sections from 8 cases (i.e., in 19% of all 43 cases examined). However, on using a digoxygenin detection system, no reactivity was found in these normal cells. An immunoperoxidase stain for chromogranin A showed that these apparently 'EBER positive' cells corresponded to normal chromogranin positive neuroendocrine cells within the gastric and duodenal mucosa. We conclude that EBV infection occurred only in the lymphoepithelioma type of gastric carcinoma and was absent from the other adenocarcinomas and from normal and benign tissues. The occasional EBER positive reaction encountered in normal cells was probably the result of a false signal arising from neuroendocrine cells as a consequence of the biotin-containing detection system.

Clinical implications of urokinase and tissue type plasminogen activators and their inhibitor (PAI-1) in breast cancer tissue.

Cytosol of primary breast cancers from 217 women of predominantly Arab ethnicity were assayed for uPA, tPA, PAI-1 and a subset for ER, PR and pS2. Serum levels of CEA and CA153 were determined during follow-up. Only tPA correlated to nodal status and tumour grade, and PAI-1 to clinical stage. PAI-1 was related to uPA and both were inversely correlated with PR and pS2 (PAI-1 also to ER). Conversely tPA was directly correlated with ER, PR and pS2. Women with high tumour uPA and PAI-1, but not tPA, had shorter overall, and relapse-free, survival. Only nodal status and clinical stage were independent predictors in multivariate analysis. However, uPA and PAI-1 were more prognostically informative than ER or PR and their usefulness may extend to delineation of patients likely to respond to adjuvant therapy.

Frequent microsatellite alterations in a predominantly younger age of onset breast cancer population.

Microsatellite instability (MSI) and loss of heterozygosity (LOH) was investigated in paired tumour and normal tissue DNA from 108 predominantly premenopausal breast cancer patients (under age 45 years at presentation) for 25 simple repeat loci interspersed across 11 chromosomes. MSI was observed at a single locus in 69 (64%) patients; 41 of these had instability at more than one site. Greatest frequency of MSI was at loci D2S1356 (33%), D2S2739 (22%), D3S1766 (21%) and D13S796 (20%). LOH was seen at a single site in 55% of patients and at two or more sites in 27 patients with greatest frequency at D2S1356 (33%), D2S443 (19%) and D17S1299 (18%). Both mutations were found in the same patient but at different loci. Clearly, choice of loci is a determining factor in assessing genomic instability. The relatively high frequency of MSI may also reflect peculiarities of this younger patient population. Occurrence of MSI or LOH was unrelated to clinical stage, nodal status, tumour size or grade or steroid receptor status. It was independent of mutations detected in exons 5-9 of the p53 gene. There was no significant association with survival. The lack of such correlations reflects a random disabling mechanism that may equally affect genes promoting cell death as well as growth.

Mechanisms of drug resistance in cancer chemotherapy.

The management of cancer involves procedures, which include surgery, radiotherapy and chemotherapy. Development of chemoresistance is a persistent problem during the treatment of local and disseminated disease. A plethora of cytotoxic drugs that selectively, but not exclusively, target actively proliferating cells include such diverse groups as DNA alkylating agents, antimetabolites, intercalating agents and mitotic inhibitors. Resistance constitutes a lack of response to drug-induced tumour growth inhibition; it may be inherent in a subpopulation of heterogeneous cancer cells or be acquired as a cellular response to drug exposure. Resistance varies. Although regulatory approval may require efficacy in as few as 20% of trial cohorts, a drug may subsequently be used in unselected patients displaying resistance to the treatment. Principal mechanisms may include altered membrane transport involving the P-glycoprotein product of the multidrug resistance (MDR) gene as well as other associated proteins, altered target enzyme (e.g. mutated topoisomerase II), decreased drug activation, increased drug degradation due to altered expression of drug-metabolising enzymes, drug inactivation due to conjugation with increased glutathione, subcellular redistribution, drug interaction, enhanced DNA repair and failure to apoptose as a result of mutated cell cycle proteins such as p53. Attempts to overcome resistance mainly involve the use of combination drug therapy using different classes of drugs with minimally overlapping toxicities to allow maximal dosages and with narrowest cycle intervals, necessary for bone marrow recovery. Adjuvant therapy with P-glycoprotein inhibitors and, in specific instances, the use of growth factor and protein kinase C inhibitors are newer experimental approaches that may also prove effective in abrogating or delaying onset of resistance. Gene knockout using antisense molecules may be another effective way of blocking drug resistance genes. Conversely, drug resistance may also be used to good purpose by transplanting retrovirally transformed CD34 cells expressing the MDR gene to protect the bone marrow during high-dose chemotherapy.

Homocholine and short-chain N-alkyl choline analogues as substrates for Torpedo choline acetyltransferase.

The kinetic parameters, Km and Vmax, for the acetylation of choline and several close analogues were determined by using (a) purified choline acetyltransferase and (b) a hypotonically lysed synaptosomal extract prepared from the electric organ of Torpedo marmorata. Whereas the Km for choline was similar in both cases (0.51 and 0.42 mM), the crude enzyme showed a three- to fivefold greater affinity for its analogues than the purified enzyme, the activity decreasing rapidly with increased N-alkyl substitution. Homocholine was a poor substrate, but was clearly acetylated by both preparations. The effect of salt on analogue acetylation by the crude enzyme was studied by increasing NaCl concentration from zero to 150 mM. There was an increase in both Km and Vmax for all substrates: choline, N,N,N-dimethylmonothylaminoethanol, -monomethyldiethylaminoethanol and -dimethylmonobutylaminoethanol showed the greatest changes, whilst N,N,N-triethylaminoethanol and -dimethylmonopropylaminoethanol and homocholine were much less affected However, in all cases, the kinetic parameter Vmax/Km remained unchanged. The maximal velocities of the different substrates varied more under conditions of high than of low salt. Sodium chloride up to 300 mM had no effect on the amount of enzyme which was bound to membranes in the synaptosomal extract. It is concluded that choline acetyltransferase has a high degree of substrate specificity, which is slightly altered by purification. The effects of salt cannot be explained as a consequence of nonspecific ionic association with membranes.