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Biomedical subjects

R C Coombes

Publications and source records attributed to R C Coombes.

At least 73 records · Page 4Linked to original sources
PubMed1997

Separated human breast epithelial and myoepithelial cells have different growth factor requirements in vitro but can reconstitute normal breast lobuloalveolar structure.

In order to investigate the specific factors controlling the growth of normal breast cell types, purified populations of human breast epithelial and myoepithelial cells from reduction mammoplasties were grown in primary culture in three defined media and their response to foetal calf serum (FCS), epidermal growth factor (EGF) and basic fibroblast growth factor (FGF2) measured using MTT growth assays. Epithelial and myoepithelial cells differed markedly in their growth requirements. Whereas epithelial cell survival was dependent on the presence of FCS, myoepithelial cell growth was dramatically inhibited by serum. EGF and FGF2 were mitogenic for epithelial cells but not myoepithelial cells, the addition of insulin being the only essential supplement required for myoepithelial cell growth. Heparin inhibited FGF2-stimulated epithelial cell growth but also basal myoepithelial cell proliferation and this inhibition could be overcome by the addition of EGF. Neutralizing antibodies to EGF also inhibited basal myoepithelial cell growth. This suggests the possibility of an autocrine role for a heparin-binding member of the EGF family in the growth of myoepithelial cells. Purified cells combined to form lobuloalveolar structures when incubated in a reconstituted basement membrane matrix (Matrigel) in the presence of EGF and FGF2.

Animals↗
PubMed1997

The detection of micrometastases in the peripheral blood and bone marrow of patients with breast cancer using immunohistochemistry and reverse transcriptase polymerase chain reaction for keratin 19.

The aim of this study was to determine whether reverse transcriptase polymerase chain reaction (RT-PCR) for keratin 19 (K19) provides additional information when combined with immunohistochemistry when used to detect micrometastases in blood and bone marrow in patients with primary breast cancer. We studied 78 patients with breast cancer who had no evidence of distant metastases. We collected blood and bone marrow, separated the mononuclear fraction and carried out RT-PCR and immunohistochemistry for K19. RT-PCR was done by two 40-cycle rounds using nested primers. In initial experiments, RT-PCR was shown to be capable of detecting one tumour cell in one million normal bone marrow cells, which was at least 10 times more sensitive than immunohistochemistry, while retaining specificity. Five per cent of the peripheral blood and 22% of the bone marrow samples contained K19 positive cells by immunohistochemistry staining. Using RT-PCR, these proportions increased to 25% and 35%, respectively. This represents a significantly greater detection frequency (P < 0.001 and P = 0.03, respectively). RT-PCR for K19 is a more sensitive method for detecting micrometastases in patients with primary breast cancer when compared with immunohistochemistry.

Adult↗
PubMed1997

Expression of keratinocyte growth factor and its receptor in human breast cancer.

The level of expression of keratinocyte growth factor (KGF) mRNA has been measured in human breast cell lines, purified populations of epithelial cells, myoepithelial cells and fibroblasts from reduction mammoplasty tissue and a panel of 42 breast cancers and 30 non-malignant human breast tissues using a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) procedure. We found similar levels of KGF mRNA in malignant and non-malignant breast tissues. The study of the amount of KGF mRNA in breast cell lines and purified populations of cells revealed that fibroblasts are the predominant source of KGF with malignant and non-malignant epithelial cells containing very low levels of KGF mRNA. We have examined the distribution of fibroblast growth factor receptor (FGFR)-2-IIIb, which is a high-affinity receptor for KGF and find that it is present on malignant and non-malignant epithelial cells. The level of FGFR-2-IIIb present on breast cancer cell lines was sufficient for KGF stimulation of breast cancer cell proliferation. Other members of the fibroblast growth factor family have been either not expressed in the human breast (FGF3, FGF4) or have been found at much reduced levels in breast cancer (FGF1, FGF2) and this is the first member of the family to potentially influence the progression of breast cancer through stimulation of cell division.

Breast↗
PubMed1997

The location of acidic fibroblast growth factor in the breast is dependent on the activity of proteases present in breast cancer tissue.

Acidic fibroblast growth factor (FGF1) and two of its receptors, FGFR1 and FGFR4, were localized in cryostat sections of normal, benign and malignant human breast tissue by immunohistochemistry. Without pretreatment, FGF1 staining was mainly seen in normal epithelial cells. However, polymerase chain reaction (PCR) analysis and immunoblotting of isolated normal epithelial and myoepithelial cells showed FGF1 mRNA and protein to be present in both cell types. Following incubation of frozen sections at 37 degrees C in phosphate-buffered saline, FGF1 staining was also revealed in myoepithelial cells and basement membrane adjacent to carcinoma cells. Treatment with protease inhibitors demonstrated that this effect was due to the activity of an endogenous protease. In contrast, FGF1 staining was found to be associated with the stroma adjacent to malignant cells only in the presence of protease inhibitors. FGFR1 and FGFR4 immunostaining was localized to both normal and malignant epithelial cells and to a lesser extent to myoepithelial cells. There was no difference in the staining intensity for the FGF receptors between normal and cancer samples. The change in location of FGF1 between normal and malignant tissues and the sensitivity of stored FGF1 to the action of endogenous proteases raises the possibility of both autocrine and paracrine roles for FGF1 in the normal and malignant human breast.

Animals↗
PubMed1997

Idoxifene is equipotent to tamoxifen in inhibiting mammary carcinogenesis but forms lower levels of hepatic DNA adducts.

Tamoxifen is an effective agent preventing mammary carcinogenesis in rats but causing liver tumours. Idoxifene is a more potent antioestrogen and is effective in patients with advanced breast cancer. We therefore compared the effects of idoxifene with tamoxifen on mammary carcinogenesis and hepatic DNA adduct formation. To do this, we undertook a study designed to compare tamoxifen with idoxifene as a chemopreventive agent in rats inoculated with N-methylnitrosourea (MNU) and also measured hepatic adduct formation. We examined the time to mammary tumour development in 272 female Ludwig/Wistar/Olac rats treated with MNU followed by tamoxifen (5 mg kg(-1)), equimolar idoxifene or vehicle three times a week for up to 24 weeks. To determine duration of effect, a second study was carried out whereby all of the 129 animals surviving at the end of treatment were entered into a surveillance programme for 27 weeks after the end of the administration period. Hepatic DNA adduct formation was examined by 32P-postlabelling in a group of rats after 24 weeks' treatment. In the first study, both idoxifene and tamoxifen were effective in preventing tumour growth as only 2 out of 21 (10%) MNU and vehicle-treated animals were alive and tumour free after 24 weeks compared with 13 out of 22 (59%) animals receiving MNU followed by idoxifene or tamoxifen (P < 0.001). The second study showed that, in both idoxifene- and tamoxifen-treated animals, a progressive regrowth of tumours occurred after cessation of therapy, as by the end of the observation period only four idoxifene-treated animals and one tamoxifen-treated animal were free from disease. In the subset of animals tested, tamoxifen-treated animals had approximately 100 times higher levels of DNA hepatic adducts than idoxifene-treated animals. Adducts were not seen in the control group. These results indicate that idoxifene is as effective a chemopreventive agent as tamoxifen in the rat while causing only very low levels of DNA adducts in liver tissue and suggest that idoxifene may be a well-tolerated chemopreventive agent in women who are at increased risk of breast cancer.

Animals↗
PubMed1997

Fibroblast growth factor 2 in breast cancer: occurrence and prognostic significance.

This paper examines the expression of fibroblast growth factor 2 (FGF-2) in the malignant human breast. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess the level of expression of FGF-2 in a series of 51 patients clinically followed up for a median of 84 months (Luqmani et al, 1992). Immunohistochemistry and Western blotting were used to show that the level of FGF-2 in breast tissues correlated with the amount of FGF-2 mRNA. FGF-2 was present in both malignant and non-malignant breast, although less was expressed in malignant tissues as determined by all three methods. Immunohistochemistry on frozen sections of breast tissue showed expression of FGF-2 in myoepithelial and epithelial cells in non-malignant samples and generally lower or undetectable levels of staining in malignant epithelial cells. The results obtained by immunohistochemistry correlated well with RT-PCR data showing similar levels of FGF-2 and FGF-2 mRNA expression in samples. No correlation was found between FGF-2 mRNA expression and T stage, nodal status or oestrogen receptor status. However, Kaplan-Meier survival plots show that higher levels of FGF-2 are associated with improved overall and disease-free survival. We suggest that FGF-2 expression may have value as a prognostic indicator in breast cancer.

Adult↗
PubMed1997

Down-regulation of a novel form of fibroblast growth factor receptor 1 in human breast cancer.

Monoclonal antibodies against two epitopes of FGFR-1 have been used to investigate FGFR-1 expression in the normal and neoplastic human breast. Different forms are detected in the different cell types constituting the normal breast. Moreover, breast cancer cells lack one form of FGFR-1. Western blot analysis showed 115-kDa and 106-kDa forms of FGFR-1 within the human breast. The 115-kDa band corresponds to the beta form of FGFR-1, whereas the 106-kDa band is truncated at the carboxyl terminus. The 106-kDa form of FGFR-1 is the major form present in breast fibroblasts and myoepithelial cells, whereas epithelial cells contain equal amounts of the 115-kDa and 106-kDa forms. Breast cancer cells, however, appear to contain only the 115-kDa form of FGFR-1. This expression pattern is reflected in malignant and non-malignant tissue samples. Using reverse transcription polymerase chain reaction (RT-PCR) analysis, we have shown that the 106-kDa FGFR-1 isoform is not the previously described alpha 2 receptor that arises from a 25-base pair insertion in the second kinase domain. It is probable that the 106-kDa FGFR-1 has different signalling properties to the full-length receptor, having lost at least one tyrosine at amino acid 766, which is required for phospholipase C activation. This form of FGFR-1 appears to be lost in all breast cancer cells analysed and its absence may have a bearing on malignancy.

Amino Acid Sequence↗
PubMed1996

Inhibitory effects of activin on the growth and morpholgenesis of primary and transformed mammary epithelial cells.

Activin is a member of the transforming growth factor beta superfamily, which is known to have activities involved in regulating differentiation and development. By using reverse transcription-PCR analysis on immunoaffinity-purified human breast cells, we have found that activin beta a and activin type II receptor are expressed by myoepithelial cells, whereas no expression was detected in other breast cell types. In examining 15 breast cell lines, we have found only four (HBL-100, MCF10-A, PMC-42, and BT 20) to be positive for activin beta a mRNA, whereas all expressed the activin type II receptor. Furthermore, we have found activin A to be a potent growth inhibitor of MCF- 7 cells (at 2 ng/ml), where it causes an arrest in G(1). Activin A does not appear to have an effect on the cell cycle of primary myoepithelial or luminal cells. However, we demonstrate that activin is an inhibitor of tubule formation by human mammary organoids in vitro. These are the first observations of activin and activin receptor in the normal human breast and in human breast cell lines and suggest a role for activin in mammary cell growth and morphogenesis.

Activins↗
PubMed1996

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.

Adult↗
PubMed1996

Multiple forms of TGF-beta 1 in breast tissues: a biologically active form of the small latent complex of TGF-beta 1.

The aim of this study was to investigate whether breast cancer growth in vivo could be due to a failure in the activation of TGF-beta 1, a growth factor which has been shown to affect the development of normal breast tissue. Tissue samples of 40 breast carcinomas and the normal adjacent tissue from 37 (henceforth referred to as 'adjacent tissue'), as well as 13 specimens of benign lesions, were included in this study. The specimens were used in vitro to produce conditioned medium (CM), and this was examined for TGF-beta 1 activity by measuring growth inhibition of the mink lung epithelial cell line CCL-64. Immunoblotting and electrophoresis were used to detect the presence of TGF-beta 1 in CM and homogenised tissue samples. We demonstrated that the majority of TGF-beta 1 in breast cancer conditioned medium was biologically active, in direct contrast to CM prepared from benign disease specimens. Furthermore, active TGF-beta 1 was also identified in CM prepared from adjacent tissue, suggesting an important early role for this growth factor in the spread of this disease. Three distinct breast cancer related (BCR) molecular weight species of TGF-beta 1 (12.5/25 kDa, 50 kDa and 95 kDa) were identified. Both the 50 kDa and 95 kDa bands immunoprecipitated by an anti-TGF-beta 1 antibody were also immunoreactive with anti-TGF-beta 1 binding protein antibodies suggesting that the 50 kDa band may comprise at least part of the previously described small latent complex of TGF-beta 1. However, using the CCL-64 cell assay, we were able to demonstrate that the 50 kDa TGF-beta 1 BCR protein was biologically active whereas the large (95 kDa) TGF-beta 1 BCR latent complex protein was not. Adjacent tissue was more likely to contain the 50 kDa form than the tumour tissues (P < 0.05). Similarly, the 50 kDa molecule was also more common in patients who had oestrogen receptor (ER) negative tumours (compared with ER positive patients; P < 0.05) and in those who had received tamoxifen treatment prior to surgery (P < 0.01). In all of these cases, the increase in the incidence of the small active complex form was accompanied by a decrease in the incidence of the high molecular weight complex (95 kDa). We confirmed that, in vitro, the 95 kDa TGF-beta 1 BCR can be proteolytically cleaved to yield a 50 kDa TGF-beta 1 BCR. Finally, we observed a correlation between the presence of the 50 kDa complex protein and reduced levels of plasminogen activator (PA), which was significant in ER negative patients (P < 0.05) and tamoxifen-pretreated patients (P < 0.01). This suggests that the secretion of this active TGF-beta 1 protein may provide breast tumours with a mechanism whereby they can escape oestrogen dependence, and may provide an explanation for the common problem of tamoxifen resistance.

Antineoplastic Agents, Hormonal↗
PubMed1996

A pilot study to evaluate paclitaxel (Taxol) as primary medical treatment for patients with inoperable stage III and IV breast carcinoma.

The activity of paclitaxel has been extensively investigated in previously treated patients with metastatic breast carcinoma. We evaluated the activity of paclitaxel as primary medical therapy in patients with stage III and IV breast carcinoma, 6 female patients were recruited with no previous history of surgery, radiotherapy or chemotherapy. Paclitaxel was administered as a 3-h infusion at a dose of 225 mg/m2 repeated every 3 weeks weekly to a maximum of 10 cycles. 2 patients achieved a complete response, one of whom had a normal trucut biopsy of the affected breast 6 months after discontinuation of chemotherapy and radiotherapy and a normal mammogram at 17 months. 3 patients achieved a partial response and one stabilised. The patients received between four and ten cycles of chemotherapy. Paclitaxel at this dose was associated with toxicity including alopecia, stomatitis, nausea and diarrhoea. Moderately severe neutropenia occurred in 4 patients, 2 requiring antibiotics but was of short duration and did not necessitate a dose reduction for subsequent courses. Paclitaxel has shown activity as primary medical therapy in patients with inoperable breast carcinoma at presentation at this dosage and schedule. One patient achieved a complete response and avoided surgery altogether and all 6 patients had their primary tumour downgraded. It may be indicated as a single agent in this context or in combination with other drugs with proven activity in breast carcinoma.

Adult↗
PubMed1996

Assesssment of the effect of pretreatment with neoadjuvant therapy on primary breast cancer.

Patients with invasive cancer of the breast (T1-4, N0-2, M0) were assigned to pretreatment based on oestrogen receptor (ER) status; patients with ER-negative tumours received chemotherapy [mitozantrone, methotrexate and mitomycin C (MMM)] for 3 months, patients with ER-positive tumours underwent endocrine therapy [luteinising hormone releasing hormone (LHRH) agonist goserelin (leuprolide-premenopausal) or 4-hydroxyandrostenedione (formestane-post-menopausal)] for 3 months. Of the first 100 patients assessed at 3 months, 47 with ER-positive tumours had a 40.4% response (premenopausal 53.8%; post-menopausal 35%) and 53 with ER-negative tumours had a 60% response (premenopausal 57%; post-menopausal 63%). Patients with early breast cancer (T1/T2) had a complete clinical resolution in 41% (16/39) of cases after MMM and in 20% (7/35) of cases following endocrine therapy compared with 14% (2/14) advanced tumours (T3/T4) following MMM and (0/12) following endocrine therapy. However, in those patients achieving a complete clinical response, subsequent appropriate surgery showed that 16 of 19 patients (84%) had evidence of residual viable tumour on histological examination.

Adult↗
PubMed1996

Keratin 19 mRNA measurement to detect micrometastases in lymph nodes in breast cancer patients.

We have used polymerase chain reaction (PCR) to measure keratin 19 mRNA in order to detect breast cancer cells invading axillary lymph nodes. In a consecutive series of 125 patients with primary breast cancer, 75 patients had no evidence of lymph node involvement by conventional histology. A total of 530 lymph nodes from these patients were examined and 106 (20%) gave a keratin 19 product detectable by Southern hybridisation. This correlated with primary tumour size (P<0.001). These 106 nodes came from 23 patients. Thus, using this technique, 23/75 (30.6%) patients were found to have evidence of lymph node involvement who would otherwise have been designated lymph node negative.

Adult↗
PubMed1996

Therapeutic effects of the aromatase inhibitor fadrozole hydrochloride in advanced breast cancer.

The endocrine and therapeutic effects of the aromatase inhibitor fadrozole hydrochloride have been assessed in 80 post-menopausal patients with recurrent breast cancer after tamoxifen failure. Treatment allocation was randomly 0.5, 1.0 or 2.0 mg orally b.d. Eight patients were not assessable for response. All patients were evaluated for toxicity (intent-to-treat analysis). In general, the patients' characteristics were well balanced between the three randomised groups. The endocrine data from this study previously reported suggest a dose-related suppression of oestrone, but not oestradiol or oestrone sulphate. The objective response rate was 17% (95% CI 8.9-27.3%) with no complete responders. Fifteen patients (21%) had stable disease (NC) and 45 patients (63%) had progressive disease (PD). The median duration of objective response was 36 weeks. The median time to treatment failure was 12.7 weeks. The log-rank test showed no statistical difference between the dosage groups. The main adverse events reported were mild to moderate severity: nausea in 11 patients (15%), hot flashes in four (5%) and somnolence in three (4%). No serious adverse events were reported. In conclusion, fadrozole is a clinically active aromatase inhibitor with a low incidence of side-effects and phase III clinical trials in post-menopausal women are currently under way.

Adult↗
PubMed1996

Adjuvant cyclophosphamide, methotrexate, and fluorouracil versus fluorouracil, epirubicin, and cyclophosphamide chemotherapy in premenopausal women with axillary node-positive operable breast cancer: results of a randomized trial. The International Collaborative Cancer Group.

PURPOSE: To determine whether a combination chemotherapy regimen that contains epirubicin (fluorouracil, epirubicin, and cyclophosphamide [FEC]) is superior to the standard cyclophosphamide, methotrexate, and fluorouracil (CMF) combination in premenopausal women with axillary node-positive operable breast cancer. PATIENTS AND METHODS: The International Collaborative Cancer Group (ICCG) conducted a large randomized trial in which two alternative schedules were used according to participating center: CMF1 versus FEC1 and CMF2 versus FEC2. RESULTS: Seven hundred fifty-nine patients were entered onto the trial. At a median follow-up time of 4.5 years, no significant benefit for the anthracycline-containing regimen was observed in terms of relapse-free (P = .61) or overall survival (P = .13). FEC1 and CMF1 appear to be of similar efficacy, but there is a suggestion that FEC2 may be superior to CMF2, since patients who received FEC2 had improved overall (P = .02) and relapse-free survival (P = .03) rates. Nausea and vomiting and alopecia were more common in the epirubicin-containing regimen (P = .001). CONCLUSION: We conclude that the FEC2 regimen, in which epirubicin replaced the methotrexate in CMF, is the preferable adjuvant chemotherapy regimen for premenopausal patients with operable axillary node-positive breast cancer.

Adult↗
PubMed1996

Identification and characterization of limonene metabolites in patients with advanced cancer by liquid chromatography/mass spectrometry.

Limonene is a farnesyl transferase inhibitor that has shown antitumor properties. The drug had been given orally to cancer patients. Plasma and urine samples collected from the patients were examined by reversed-phase HPLC-atmospheric pressure chemical ionization and electrospray ionization MS. The drug underwent rapid conversion to hydroxylated and carboxylated derivatives. Characterization and structural elucidation of the metabolites were achieved by LC/MS and NMR. Five major metabolites were detected in the plasma extracts, namely limonene-1,2-diol, limonene-8,9-diol, perillic acid, an isomer of perillic acid, and dihydroperillic acid. Urinary metabolites comprised the glucuronides of the two isomers of perillic acid, dihydroperillic acid, limonene-8,9-diol, and a monohydroxylated limonene.

Adult↗
PubMed1995

Fibroblast growth factor receptors (FGFRs) localize in different cellular compartments. A splice variant of FGFR-3 localizes to the nucleus.

We have raised specific antibodies to the second immunoglobulin-like domain of fibroblast growth factor receptors (FGFRs) and used these to investigate the expression and subcellular localization of FGFR-1, -2, -3, and -4 in breast epithelial cells. All four receptors classes could be detected in breast cell lines; however, FGFR-4 and FGFR-2 appeared to be expressed at a higher level in breast cancer cell lines than in normal epithelial cells. Surprisingly, FGFR-3 localized in the cell nucleus by immunofluorescence. A second antibody to a separate epitope confirmed this finding and showed that the form of FGFR-3 present must contain an intact kinase domain as well as the growth factor binding domain. Western analysis of fractionated cells revealed the presence of two forms of FGFR-3 of 135 and 110 kDa. The 110-kDa form was predominantly found in the nucleus, whereas the 135 kDa form was sometimes found in the nucleus. RT-PCR analysis of FGFR-3 mRNA showed the presence of a splice variant in which exons 7 and 8 are deleted. This results in the translation of FGFR-3 missing the transmembrane domain but with an intact kinase domain, which could be a soluble, intracellular receptor. Transfection experiments showed that FGFR-3 containing this deletion and no signal peptide gave an identical nuclear staining pattern to that seen in breast epithelial cells. We conclude that two forms of FGFR-3 are present in breast epithelial cells; a full-length 135-kDa receptor, which has a conventional membrane localization, and a novel soluble form of 110 kDa.

Alternative Splicing↗