Computer‐aided design and physiological testing of a luteinising hormone‐releasing hormone analogue for ‘adjuvant‐free’ immunocastration

Summary A variant form of the human oestrogen receptor (ER) mRNA lacking sequences encoded within exon 5 has been described (Fuqua SAW, Fitzgerald SD, Chamness GC, Tandon AK, McDonnell DP, Nawaz Z, O'Malloy BW, McGuire WL 1991, Cancer Res 51: 105-1 09).We have examined the expression of the exon 5-deleted ER (HEA5) mRNA variant in breast biopsies using reverse transcriptase polymerase chain reaction (RT -PCR). HEA5 mRNA was present in only 13% of non-malignant breast tissues compared with 32% of carcinomas (95% Cl, P=0.05). Presence of the HEA5 mRNA was associated with the presence of immunohistochemically detected ER (P=0.015) and progesterone receptor (PR) (P=0.02). There was a positive correlation between the presence of HEA5 and disease-free survival (P=0.05), suggesting that the presence of HEA5 may be an indicator of better prognosis. We have raised a monoclonal antibody specific to the C-terminal amino acids of HEA5. This antibody recognized the variant but not the wild-type ER protein. We show that HEA5 protein is present in breast cancer using immunohistochemical techniques. We also analysed trans-activation by HEA5 in mammalian cells and showed that, in MCF-7 cells, HEA5 competes with wild-type ER to inhibit ERE-dependent trans-activation. Our results indicate that this variant is unlikely to be responsible for endocrine resistance of breast cancer, but its presence at both the mRNA and protein level suggest that it may, nevertheless, be involved in regulating the expression of oestrogen-responsive genes in breast cancer.Keywords: breast neoplasm; exon; polymerase chain reaction; oestrogen receptor; transcription Two-thirds of human breast carcinomas are characterized by the presence of appreciable amounts of oestrogen receptor (ER) protein.A proportion of these tumours also contain progesterone receptor (PR) and it is generally accepted that ER regulates PR gene expression. The presence of ER is correlated with a better prognosis and ER+/PR+ tumours are much more likely to respond to endocrine therapy than ER-/PR-tumours. Interestingly, ER-/PR+ tumours are twice as likely to respond as ER+/PR-tumours. A significant proportion of ER+/PR+ tumours, however, fail to respond to endocrine therapy and those that do so eventually become resistant to such therapy. The mechanisms leading to endocrine resistance are not yet clear (for reviews see McGuire, 1978;McGuire et al, 1991;Fuqua, 1994;Horwitz, 1994;Sluyser, 1994).The human oestrogen receptor cDNA and its gene (Ponglikitmongkol et al, 1988) have been cloned and the molecular mechanisms by which it acts are well understood. Alignment of the predicted ER amino acid sequences from different species shows that it can be divided into six regions A to F on the basis of differing amino acid sequence homology . Functional studies have shown that region C encodes the DNA- Kumar et al, 1987). Regions A/B and E contain trans-activation functions 1 (AFI) and 2 (AF-2) respectively (Kumar et al, , 1987Webster et al, 1988; Lees et al, 1989; Tora et al, 1989a;Berry et ...

Section: Statistical Analysesmentioning

confidence: 99%

Presence of exon 5-deleted oestrogen receptor in human breast cancer: functional analysis and clinical significance

Desai

Luqmani²,

Walters³

et al. 1997

“…The peptides were prepared using a NovaSyn Crystal automated peptide synthesizer on a KA (Kieselguhr/polydimethyVacrylamide) resin (Calbiochem Novabiochem) and peptide purity was checked by reverse-phase high-performance liquid chromatography (HPLC). The peptides were coupled to a purified protein derivative of tuberculin (PPD) (Morrison et al, 1987) and were then injected into female Balb/c mice. The spleen, from a selected mouse, was removed and the splenocytes fused with Sp2/O myeloma cells as previously described (Galfre and Milstein, 1981).…”

mentioning

confidence: 99%

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

Coope

Browne²,

Yiangou³

et al. 1997

Summary 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 370C 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.Keywords: breast cancer; fibroblast growth factor 1; protease; immunohistochemistry Fibroblast growth factor 1 (FGF1) belongs to a family of multifunctional polypeptides that are involved in a wide array of biological processes, which include cellular proliferation and differentiation, angiogenesis, chemotaxis, embryonal development and tissue repair (Basilico and Moscatelli, 1992). To date, the FGFs consist of a family of nine homologous polypeptide growth factors that include FGF1 (acidic FGF), FGF2 (basic FGF), FGF3 (int-2), FGF4 (hst-l/Kaposi FGF), FGF5, FGF6 (hst-2), FGF7 (keratinocyte growth factor), FGF8 (androgen-induced growth factor) and FGF9 (glial-activating factor) (Basilico and Moscatelli, 1992;Tanaka et al, 1992;Miyamoto et al, 1993). These proteins share 35-50% overall homology of their amino acid sequences (Basilico and Moscatelli, 1992;Givol and Yayon, 1992).Unlike other members of the family, FGF1, FGF2 and FGF9 are synthesized without a signal peptide sequence and thus may remain sequestered in the cell (Basilico and Moscatelli, 1992;Cao and Pettersson, 1993). However, release of FGF may occur through leakage from damaged cells or from viable cells via a novel mechanism (Mignatti et al, 1992;Cao and Pettersson, 1993). Yeoman (1993) has postulated that proteoglycan-bound FGF may be released from the cell surface or extracellular matrix by the action of proteases, and Briozzo et al (1991) have shown that Received 10 June 1996 Revised 26 November 1996 Accepted 3 December 1996 Correspondence to: J Gomm MCF7 breast cancer cells secrete cathepsin D, which is able to digest the extracellular matrix and releas...

“…The peptide was prepared on a Wang resin using the AMS 422 Multiple Peptide Synthesizer by the Fmoc method, and its purity was checked by reverse-phase HPLC. Ten milligrams of the peptide was coupled to 10 mg of purified protein derivative (Morrison et al, 1987), and this conjugate was used to inject female Balb/c mice. The splenocytes of one of the mice were fused with Sp2/0 myeloma cells and, after 7 days, the hybridoma supernatants were screened by ELISA.…”

Section: Antibodiesmentioning

confidence: 99%

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

Yiangou

Cox²,

Bansal³

et al. 1997

Summary 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 11 5-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 1 06-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.Keywords: fibroblast growth factor receptor 1; splice variant; human breast cancer; epithelial/myoepithelial cell; monoclonal antibodyThe family of fibroblast growth factors comprises nine structurally related polypeptides that are mitogenic for a variety of cells and that are involved in embryogenesis, angiogenesis and differentiation (Burgess and Maciag, 1989;Miyamoto et al, 1993). Their cellular response is thought to be mediated through cell surface receptors that belong to the superfamily of tyrosine kinase receptors (Johnson et al, 1991;Keegan et al, 1991;Partanen et al, 1991;Jaye et al, 1992). Extracellular matrix and cell surface heparan sulphate proteoglycans are involved in the interaction of these growth factors with their receptors (Klagsbrun and Baird, 1991;Kan et al, 1993).The four fibroblast growth factor receptors are type four tyrosine kinase receptors and are encoded by four distinct genes. They consist of an extracellular ligand binding domain, a transmembrane part and an intracellular split kinase domain that is involved in signal transduction (Figure lA). They share a high amino acid sequence homology and are characterized by a large number of variant forms generated by alternative mRNA splicing (Hou et al, 1991;Jaye et al, 1992). Variants include: the full-length alpha form; the beta form lacking the first immunoglobulin domain; the intracellular gamma form lacking a signal peptide, the first immunoglobulin domain and the acidic box; and a truncated form, alpha 2, which results from a 25-bp insertion in the second kinase domain leading to a frame shift ( Figure IB). The precise biological func...