RESUMEN
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.
Asunto(s)
Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , ARN Interferente Pequeño/farmacología , Secuencia de Bases , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido NucleicoRESUMEN
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.
Asunto(s)
Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Farmacogenética/tendencias , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Biología Molecular/tendencias , Neoplasias/patología , Proteína Quinasa C/antagonistas & inhibidores , Insuficiencia del TratamientoRESUMEN
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.
Asunto(s)
Alelos , Neoplasias de la Mama/genética , Repeticiones de Microsatélite/genética , Polimorfismo Genético , Neoplasias de la Mama/diagnóstico , Femenino , Marcadores Genéticos , HumanosRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/genética , Repeticiones de Microsatélite/genética , Adulto , Edad de Inicio , Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Femenino , Frecuencia de los Genes , Marcadores Genéticos , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Posmenopausia , PremenopausiaRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/metabolismo , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Proteínas , Activador de Tejido Plasminógeno/biosíntesis , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Antígeno Carcinoembrionario/sangre , Citosol/metabolismo , Supervivencia sin Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Persona de Mediana Edad , Mucina-1/sangre , Pronóstico , Biosíntesis de Proteínas , Receptores de Estrógenos/biosíntesis , Receptores de Progesterona/biosíntesis , Factores de Tiempo , Factor Trefoil-1 , Proteínas Supresoras de TumorRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/radioterapia , Carcinoma de Células Escamosas/etiología , Neoplasias Esofágicas/etiología , Neoplasias Inducidas por Radiación/etiología , Neoplasias Primarias Secundarias/etiología , Resultado Fatal , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Adenocarcinoma/virología , Infecciones por Virus de Epstein-Barr/virología , Gastrectomía , Herpesvirus Humano 4/aislamiento & purificación , Linfoma/virología , Neoplasias Gástricas/virología , Adenocarcinoma/diagnóstico , Adenocarcinoma/cirugía , Linfocitos B/metabolismo , Linfocitos T CD8-positivos/metabolismo , Cromogranina A , Cromograninas/metabolismo , Digoxigenina , Células Epiteliales/virología , Infecciones por Virus de Epstein-Barr/diagnóstico , Infecciones por Virus de Epstein-Barr/cirugía , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Herpesvirus Humano 4/genética , Humanos , Técnicas para Inmunoenzimas , Hibridación in Situ , Linfoma/diagnóstico , Linfoma/cirugía , Úlcera Péptica/diagnóstico , Úlcera Péptica/cirugía , Úlcera Péptica/virología , ARN Viral/genética , ARN Viral/metabolismo , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/cirugíaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Antígenos de Neoplasias/análisis , Neoplasias de la Mama/genética , Antígenos CD5/análisis , Cromosomas Humanos Par 13 , Leucemia Linfocítica Crónica de Células B/genética , Neoplasias Primarias Secundarias/genética , Anciano , Neoplasias de la Mama/química , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Neoplasias Primarias Secundarias/químicaAsunto(s)
ADN de Neoplasias/química , ADN/química , Repeticiones de Microsatélite , Oligodesoxirribonucleótidos/aislamiento & purificación , Repeticiones de Trinucleótidos , Secuencia de Bases , Mama/química , Neoplasias de la Mama/química , ADN/aislamiento & purificación , Cartilla de ADN , ADN de Neoplasias/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida/instrumentación , Electroforesis en Gel de Poliacrilamida/métodos , Femenino , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodosRESUMEN
We have analysed cytoplasmic and nuclear extracts of breast-cancer tissue from a total of 799 patients, measuring both oestrogen and progesterone receptors (ER, PR) using either the ligand binding assay (LBA) or the enzyme immunoassay technique (EIA). Mean and median receptor levels were much lower than those widely reported by others. For ER, this may in part be a consequence of the younger median age of the patient group. The frequency of positivity, using consensus cut-off values for clinical evaluation, was also lower than that reported by the EORTC Receptor Study Group. Although the measurements comparing the 2 methods were statistically correlated in terms of positivity, based on the above criteria for clinical assessment, concordance was considered to be relatively poor, particularly for ER when assayed in the same samples by the 2 methods. In cytosolic but not nuclear extracts, the LBA method gave a higher median value for ER than the EIA (except in the group that had EIA values greater than 15 fmol/mg protein); for PR, median values were higher with EIA in both cell fractions. There was an excellent correlation between receptor amounts in cytosolic and nuclear extracts for both ER and PR using the EIA; this was significantly better than with LBA. We also observed a correlation between ER and PR in both cytosolic and nuclear fractions which was most pronounced when the analysis was done by EIA. The amounts of ER in the cytosolic fraction were also correlated with the those of PR in the nuclear fraction and ER in the nuclear fraction with PR in the cytosolic fraction, but only when the EIA method was used. We conclude that the EIA method appears to be more sensitive and gives biologically more reliable results. However, the disagreement between the methods may be due to legitimate recognition of altered forms of the receptor and may be of biological significance. Although the presence of receptor in the cytosolic fraction is artifactual, its measurement by EIA does parallel the amounts of nuclear receptor, which may be a more relevant biological parameter.
Asunto(s)
Neoplasias de la Mama/química , Núcleo Celular/química , Citosol/química , Técnicas para Inmunoenzimas , Proteínas de Neoplasias/análisis , Ensayo de Unión Radioligante , Receptores de Estrógenos/análisis , Receptores de Progesterona/análisis , Adulto , Anciano , Artefactos , Neoplasias de la Mama/patología , Estudios de Evaluación como Asunto , Femenino , Humanos , Persona de Mediana Edad , Sensibilidad y EspecificidadRESUMEN
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: 105-109). We have examined the expression of the exon 5-deleted ER (HE delta5) mRNA variant in breast biopsies using reverse transcriptase polymerase chain reaction (RT - PCR). HE delta5 mRNA was present in only 13% of non-malignant breast tissues compared with 32% of carcinomas (95% CI, P=0.05). Presence of the HE delta5 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 HE delta5 and disease-free survival (P=0.05), suggesting that the presence of HE delta5 may be an indicator of better prognosis. We have raised a monoclonal antibody specific to the C-terminal amino acids of HE delta5. This antibody recognized the variant but not the wild-type ER protein. We show that HE delta5 protein is present in breast cancer using immunohistochemical techniques. We also analysed trans-activation by HE delta5 in mammalian cells and showed that, in MCF-7 cells, HE delta5 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.
Asunto(s)
Neoplasias de la Mama/metabolismo , Exones , Regulación Neoplásica de la Expresión Génica , Receptores de Estrógenos/biosíntesis , Eliminación de Secuencia/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/biosíntesis , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Células COS , Cartilla de ADN/química , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , ARN Mensajero/biosíntesis , Receptores de Estrógenos/genética , Receptores de Estrógenos/inmunología , Receptores de Progesterona/biosíntesis , Análisis de Supervivencia , Transfección/genética , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma/metabolismo , Factor 2 de Crecimiento de Fibroblastos/biosíntesis , Adulto , Anciano , Western Blotting , Mama/química , Mama/metabolismo , Neoplasias de la Mama/química , Neoplasias de la Mama/diagnóstico , Carcinoma/química , Carcinoma/diagnóstico , Carcinoma Ductal de Mama/química , Carcinoma Ductal de Mama/diagnóstico , Supervivencia sin Enfermedad , Femenino , Factor 2 de Crecimiento de Fibroblastos/análisis , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Pronóstico , ARN Mensajero/análisis , ARN Neoplásico/química , Análisis de SupervivenciaRESUMEN
In this study, we used immunohistochemical and biochemical analysis to show that gp200-MR6, a 200 kDa molecule that is functionally associated with the human interleukin 4 (IL-4) receptor complex, is expressed at high levels on normal breast epithelial tissues, at lower levels on in situ carcinomas, and that the expression is lost in the invasive carcinoma of the breast. Furthermore, a preliminary study showed that benign epithelial hyperplasia of the breast expresses the gp200-MR6 heterogeneously. Two populations of cells have been observed: MR6 positive and MR6 negative. Interestingly, MR6-positive cells were observed to have different morphology from those that were MR6 negative; the nuclei of the former were larger and rounded in shape, whereas the nuclei of the latter were relatively small and oval in shape. In sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, monoclonal antibody MR6 detects the same molecular weight molecule in both normal and transformed tissue, indicating that the molecule is not a product of a truncated gene. The intensity of the gp200-MR6 bands correlates with the immunohistochemical data, indicating that the molecule is expressed at high levels in normal tissue and at lower levels in malignant tissue. These results suggest that analysis of gp200-MR6 expression may be useful in tumour grading and prognostic evaluation in breast cancer. Moreover, the molecule may be involved early in the process of tumorigenesis of the breast, in which a loss or a down-regulation of gp200-MR6 could contribute towards tumour development and progression via an effect on cell growth and differentiation.
Asunto(s)
Antígenos CD , Neoplasias de la Mama/química , Mama/química , Carcinoma in Situ/química , Glicoproteínas/análisis , Lectinas Tipo C , Proteínas de Neoplasias/análisis , Receptores de Superficie Celular , Western Blotting , Mama/patología , Carcinoma/química , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Hiperplasia , Antígenos de Histocompatibilidad MenorRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/genética , Factor 2 de Crecimiento de Fibroblastos , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Transcripción Genética , Adulto , Anciano , Anciano de 80 o más Años , Empalme Alternativo , Secuencia de Bases , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células Cultivadas/metabolismo , ADN , Femenino , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Células Tumorales Cultivadas/metabolismoRESUMEN
We have measured the amount of fibroblast growth factor 1 (FGF-1) mRNA and protein in primary breast cancers and non-malignant breast tissue and have found greatly reduced levels in breast cancer compared with non-malignant tissue. A total of 116 breast cancers and 37 biopsies taken from non-malignant breast were compared for FGF-1 mRNA expression using reverse transcriptase-polymerase chain reaction (RT-PCR) and significantly lower levels were found in the cancer tissues (P < 0.001). These findings were confirmed at the protein level where four out of five breast cancers contained no detectable FGF-1 and a fifth cancer had a low level of FGF-1 compared with three samples from reduction mammoplasties. Similar results were obtained from breast cell lines in which 80% of cancer cell lines had very low levels of FGF-1, whereas all non-malignant breast cell lines contained higher levels of FGF-1. Immunohistochemical analysis indicated that FGF-1 was present in the luminal epithelial cells of the non-malignant breast but was absent from cancer cells. The decreased levels of FGF-1 in breast cancer may indicate that stimulation of cancer cells is resulting in down-regulation of FGF-1 expression or may implicate FGF-1 as a differentiation factor rather than a growth factor at its physiological concentration in the breast.
Asunto(s)
Neoplasias de la Mama/metabolismo , Mama/metabolismo , Factor 1 de Crecimiento de Fibroblastos/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Western Blotting , Mama/química , Neoplasias de la Mama/química , Línea Celular , Femenino , Factor 1 de Crecimiento de Fibroblastos/análisis , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/biosíntesis , Biosíntesis de Proteínas , Proteínas/análisis , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Valores de Referencia , Células Tumorales CultivadasRESUMEN
The expression of variant mRNAs encoding isoforms of fibroblast growth factor receptor (FGFR)1 with either 2 or 3 Ig-like loops in the extracellular domain was investigated in human breast tissues and cell lines using a polymerase chain reaction amplification method. Almost all tissues contained both forms of FGFR1, but cancers (n = 137) had a significantly lower proportion of the transcript that encoded the full 3-loop form compared with non-malignant biopsies (n = 34). This was confirmed using microdissected populations of normal and cancerous cells from frozen tissue sections. A high ratio of the 2- to 3-loop form was found to be predictive of reduced relapse-free survival. In both groups, however, the predominant form of FGFR1 was that encoding the 2-loop receptor. Cell lines derived from a variety of tissues, including breast, also co-expressed both variants of FGFR1, suggesting their presence within the same cell type. Again, there was a similar preponderance of the shorter isoform. Our results were confirmed at the protein level, where out of 5 cancers analysed 4 expressed more of the 2-loop form than the 3-loop form. Our findings suggest that cells may normally simultaneously express several splice variants of FGFR1, and aberrant expression or a change in their relative amounts (i.e., in malignancy) could contribute to modified responses to either autocrine or paracrine factors.