RESUMEN
The mammalian target of rapamycin (mTOR) plays a key role in the regulation of cellular metabolism, growth and proliferation. It forms two multi-protein complexes known as complex 1 (mTORC1) and 2 (mTORC2). Raptor and Rictor are the core proteins for mTORC1 and mTORC2, respectively. This study examines the relationship between mTORC1, Rictor and Raptor mRNA expression and human breast cancer. Furthermore, the correlation between mTORC1 and hTERT was investigated. Breast cancer tissues (n=150) and normal tissues (n=31) were analysed using reverse transcription and quantitative PCR. Transcript levels were correlated with clinicopathological data. Higher mTOR expression was noted in breast cancer tissue (P=0.0018), higher grade tumours (grade 2 vs. 3, P=0.047), in ductal tumours (P=0.0014), and was associated with worse overall survival (P=0.01). Rictor expression was significantly higher in background breast tissues compared with tumours and was inversely related to the Nottingham Prognostic Index (NPI1 vs. 2, P=0.03) and tumour grade (grade 1 vs. 3, P=0.01) and was associated with better overall (P=0.037) and disease-free survival (P=0.048). The mRNA expression of Raptor was higher in tumours compared with normal tissues. Furthermore, the expression of Raptor was associated with a higher tumour grade (grade 1 vs. 3, P=0.027). A highly significant positive correlation between mTOR and hTERT (P<0.00001) was observed. These observations are consistent with the role of mTORC1 in the anti-apoptosis pathway and suggest that selective inhibitors of mTORC1 may be more efficacious in human breast cancer. Our findings support the hypothesis that mTORC1 is an important upregulator of telomerase in breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Portadoras/biosíntesis , Complejos Multiproteicos/biosíntesis , Serina-Treonina Quinasas TOR/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/agonistas , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Supervivencia sin Enfermedad , Femenino , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Pronóstico , ARN Mensajero/genética , Proteína Asociada al mTOR Insensible a la Rapamicina , Proteína Reguladora Asociada a mTOR , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
Immortalization (senescence bypass) is a critical rate-limiting step in the malignant transformation of mammalian somatic cells. Human cells must breach at least two distinct senescence barriers to permit unfettered clonal evolution during cancer development: (1) stress- or oncogene-induced premature senescence (SIPS/OIS), mediated via the p16-Rb and/or ARF-p53-p21 tumour-suppressive pathways, and (2) replicative senescence triggered by telomere shortening. In contrast, because their telomerase is constitutively active, cells from small rodents possess only the SIPS/OIS barrier, and are therefore useful for studying SIPS/OIS bypass in isolation. Dermal fibroblasts from the Syrian hamster (SHD cells) are exceptionally resistant to spontaneous SIPS bypass, but it can be readily induced following exposure to a wide range of chemical and physical carcinogens. Here we show that a spectrum of carcinogen-specific mutational and epigenetic alterations involving the INK4A (p16), p53 and INK4B (p15) genes are associated with induced SIPS bypass. With ionizing radiation, immortalization is invariably accompanied by efficient biallelic deletion of the complete INK4/CDKN2 locus. In comparison, SHD cells immortalized by the powerful polycyclic hydrocarbon carcinogen benzo(a)pyrene display transversion point mutations in the DNA-binding domain of p53 coupled with INK4 alterations such as loss of expression of p15. Epimutational silencing of p16 is the primary event associated with immortalization by nickel, a human non-genotoxic carcinogen. As SIPS/OIS bypass is a prerequisite for the immortalization of normal diploid human epithelial cells, our results with the SHD model will provide a basis for delineating combinations of key molecular changes underpinning this important event in human carcinogenesis.
Asunto(s)
Carcinógenos/toxicidad , Senescencia Celular , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Genes p53 , Proteína p53 Supresora de Tumor/genética , Animales , Benzo(a)pireno/toxicidad , Línea Celular , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Senescencia Celular/efectos de la radiación , Cricetinae , Células Epiteliales/metabolismo , Eliminación de Gen , Humanos , Mesocricetus , Mutación , Níquel/farmacologíaRESUMEN
BACKGROUND: SET domain containing protein 2 (SETD2) is a histone methyltransferase that is involved in transcriptional elongation. We previously demonstrated SETD2 to be a potential tumour suppressor gene in breast cancer. The aim of this study was to compare SETD2 expression in breast cancer with that in adjacent non-cancerous breast tissue (ANCT) in paired samples. A hypothesis is proposed that explains the mode of action of SETD2 as a tumour suppressor gene. MATERIALS AND METHODS: Paired samples of tumour and adjacent non-cancerous tissue (ANCT) from 25 patients were analysed. The levels of transcription of SETD2 were determined using quantitative polymerase chain reaction and normalized against cytokeratin 19. Immunohistochemical staining with appropriate antibodies against SETD2 protein was also performed in selected samples. RESULTS: Levels of SETD2 mRNA were significantly higher in ANCT when compared to those in tumour samples (p=0.01). Immunohistochemistry also demonstrated a higher protein expression in ANCT. CONCLUSION: This study offers further evidence that SETD2 behaves like a tumour suppressor gene. Our hypothesis links SETD2 mode of action with telomerase regulation through human telomerase reverse transcriptase (hTERT). Several studies have emphasised the importance of histone methylation of hTERT promotor in telomerase regulation. SETD2 function of histone methylation could be the missing link in this chain which could explain the potential tumour suppressor function of SETD2.
Asunto(s)
Neoplasias de la Mama/genética , Genes Supresores de Tumor , N-Metiltransferasa de Histona-Lisina/genética , Neoplasias de la Mama/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/biosíntesis , Humanos , Inmunohistoquímica , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
UNLABELLED: DLEC1 (deleted in lung and oesophageal cancer), located on 3p22.3, is a candidate tumour suppressor gene in lung, esophageal, and renal cancer. The aim of this study was determine whether the mRNA expression levels of DLEC1 were consistent with a tumour suppressive function. MATERIALS AND METHODS: A total of 153 samples were analysed. The levels of transcription of DLEC1 were determined using quantitative PCR and normalised against (CK19). Transcript levels within breast cancer specimens were compared to normal background tissues. RESULTS: Levels of transcription were lower [corrected] in tumour samples compared to adjacent non cancerous tissue (ANCT) samples but this was not statistically significant (median 0.167 vs. 0.03; p=0.138). DLEC1 expression levels were significantly lower in samples from patients who developed metastasis, local recurrence, or died of breast cancer when compared to those who were disease free for >10 years (p=0.041). DISCUSSION: These findings are consistent with a possible tumour suppressor function of DLEC1 in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas Supresoras de Tumor/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Genes Supresores de Tumor , Humanos , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transcripción Genética , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
BACKGROUND: SET domain containing protein 2 (SETD2) is a histone methyltransferase that is involved in transcriptional elongation. There is evidence that SETD2 interacts with p53 and selectively regulates its downstream genes. Therefore, it could be implicated in the process of carcinogenesis. Furthermore, this gene is located on the short arm of chromosome 3p and we previously demonstrated that the 3p21.31 region of chromosome 3 was associated with permanent growth arrest of breast cancer cells. This region includes closely related genes namely: MYL3, CCDC12, KIF9, KLHL18 and SETD2. Based on the biological function of these genes, SETD2 is the most likely gene to play a tumour suppressor role and explain our previous findings. Our objective was to determine, using quantitative PCR, whether the mRNA expression levels of SETD2 were consistent with a tumour suppressive function in breast cancer. This is the first study in the literature to examine the direct relationship between SETD2 and breast cancer. METHODS: A total of 153 samples were analysed. The levels of transcription of SETD2 were determined using quantitative PCR and normalized against (CK19). Transcript levels within breast cancer specimens were compared to normal background tissues and analyzed against conventional pathological parameters and clinical outcome over a 10 year follow-up period. RESULTS: The levels of SETD2 mRNA were significantly lower in malignant samples (p = 0.0345) and decreased with increasing tumour stage. SETD2 expression levels were significantly lower in samples from patients who developed metastasis, local recurrence, or died of breast cancer when compared to those who were disease free for > 10 years (p = 0.041). CONCLUSION: This study demonstrates a compelling trend for SETD2 transcription levels to be lower in cancerous tissues and in patients who developed progressive disease. These findings are consistent with a possible tumour suppressor function of this gene in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Regulación Neoplásica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , Neoplasias de la Mama/metabolismo , Femenino , Estudios de Seguimiento , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Estadificación de Neoplasias , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: The E2F family of transcription factors are key regulators of genes involved in cell cycle progression, cell fate determination, DNA damage repair and apoptosis. E2F1 is unique in that it contributes both to the control of cellular proliferation and cellular death. Furthermore, unlike other E2Fs, E2F1 responds to various cellular stresses. This study aimed to examine the level of mRNA expression of E2F1 gene in normal and malignant breast tissue and correlate the level of expression to tumour stage. MATERIALS AND METHODS: One hundred and twenty-seven breast cancer tissue and 33 normal tissues were analyzed. Levels of transcription of E2F1 were determined using real-time quantitative PCR, normalized against CK19. Levels of expression were analyzed against TNM stage, nodal involvement, tumour grade and distant metastasis. RESULTS: The levels of E2F1 mRNA were lower in malignant tissues. They declined further with increasing TNM stage. This became statistically significant when TNM stages 3 and 4 were compared to TNM stages 1 and 2 disease (TNM1 vs. TNM3 p = 0.032; TNM1 vs. TNM4 p = 0.032; TNM2 vs. TNM3 p = .019; TNM2 vs. TNM4 p = 0.021). The levels of E2F1 also fell with increasing tumour grade, when comparing grade 2 and 3 with grade 1, however, the differences were not statistically significant. CONCLUSION: These results are highly suggestive of the role of E2F1 as a tumour suppressive gene in human breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Factor de Transcripción E2F1/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Factor de Transcripción E2F1/biosíntesis , Dosificación de Gen , Expresión Génica , Genes Supresores de Tumor , Humanos , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Telomerase activity has been significantly associated with nodal metastasis and cellular proliferation in human breast cancer, indicating that its degree of expression has some form of vital control over the invasive nature of the malignancy concerned. Of the telomerase subunits, the reverse transcriptase (hTERT) is the main determinant of enzyme activity. Vascular endothelial growth factors (VEGF)-C and (VEGF)-D, matrix metalloprotease type 1 (MMP-1) and protease-activated receptors (PARs) have all been linked to promotion of tumour invasiveness and metastatic dissemination. This study aims to examine the association between hTERT transcription and that of VEGF-D, VEGF-C, MMP-1, PAR1a and PAR1b through a correlative analysis of the mRNA transcripts of these genes in human breast cancer. MATERIALS AND METHODS: Breast cancer tissues (n = 116) and normal tissues (n-31) were collected immediately after surgery and stored at -80 degrees C until use. The level of hTERT transcripts from the prepared DNA from the above samples was determined using real time-quantitative PCR based on the Amplifluor technology. The levels of the transcript were generated from a standard that was simultaneously amplified with the samples. Normalisation against cytokeratin 19 (CK19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also carried out. RESULTS: There was a positive correlation between hTERT mRNA expression (after CK19 normalisation) with both VEGF-D and MMP-1 in human breast cancer. PAR1 was seen to correlate with hTERT (after GAPDH normalisation) with a highly significant correlation with PAR1a alone. However there was no correlation between hTERT transcription and VEGF-C or with PAR1b alone. CONCLUSION: Our findings suggest that hTERT is a potential up-regulator of MMP-1, PAR1 and VEGF-D expression and this may explain its apparent control over the invasiveness and metastasis of the malignancy concerned.
Asunto(s)
Neoplasias de la Mama/metabolismo , Metaloproteinasa 1 de la Matriz/biosíntesis , ARN Mensajero/biosíntesis , Telomerasa/biosíntesis , Factor C de Crecimiento Endotelial Vascular/biosíntesis , Factor D de Crecimiento Endotelial Vascular/biosíntesis , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Humanos , Estadificación de Neoplasias , ARN Mensajero/genética , Receptor PAR-1/biosíntesis , Receptor PAR-1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/genética , Transcripción Genética , Factor C de Crecimiento Endotelial Vascular/genética , Factor D de Crecimiento Endotelial Vascular/genéticaRESUMEN
BACKGROUND: Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. hTERT (human telomerase reverse transcriptase) gene is the rate-limiting determinant of telomerase reactivation. The present study aims to quantitatively measure the expression of hTERT mRNA in human breast cancer, examine the association between hTERT and the clinicopathological characteristics of the cancer specimens including the Nottingham Prognostic Index (NPI) and to explore the relationship between hTERT expression and clinical outcome. MATERIALS AND METHODS: RNA was extracted from 116 breast carcinomas and 31 matched adjacent non-cancerous tissue (ANCT). hTERT mRNA expression was estimated by reverse transcriptase-PCR (RT-PCR) and Taqman methodology. RESULTS: hTERT mRNA was present in all of the cancerous specimens (mean=0.1701, median=0.0205) and most ANCT specimens with levels being 2.6 times higher in the cancerous tissue than in ANCT (mean=0.156 vs. 0.68, p=0.18). The mean mRNA levels increased with NPI scores (0.0816 for NPI 1, 0.1186 for NPI 2 and 0.68 for NPI 3), however this failed to reach statistical significance (P-values= 0.33 for NPI 1 vs. 2, 0.27for NPI2 vs. 3 and 0.24 for NPI 1 vs. 3). hTERT levels also increased with increasing tumour's grade (mean= 0.0459 for grade 1, 0.111for grade 2, and 0.27 for grade 3) but this trend did not reach a statistical significance. Low levels of hTERT were associated with mucinous carcinoma compared with ductal (p=0.023) and lobular (p=0.021) types. hTERT mRNA levels were higher in patients who had recurrent disease or died from breast cancer compared with those who remained alive without disease after a median follow up of 6 years (p=0.0026). CONCLUSION: High hTERT mRNA levels are associated with a poor clinical outcome in human breast cancer and should be included as a prognostic marker in future validation studies.
Asunto(s)
Neoplasias de la Mama/genética , ARN Mensajero/biosíntesis , Telomerasa/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Supervivencia sin Enfermedad , Humanos , Estadificación de Neoplasias , ARN Mensajero/genética , Telomerasa/biosíntesis , Resultado del TratamientoRESUMEN
BACKGROUND: Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. The hTERT (human telomerase reverse transcriptase) subunit seems to be the rate-limiting determinant of telomerase and knowledge of factors controlling hTERT transcription may be useful in therapeutic strategies. The hTERT promoter contains binding sites for c-Myc and there is some experimental and in vitro evidence that c-Myc may increase hTERT expression. We previously reported no correlation between c-Myc mRNA expression and hTERT mRNA or telomerase activity in human breast cancer. This study aims to examine the correlation between hTERT expression as determined by immunohistochemistry and c-Myc expression, lymph node status, and tumour size and grade in human breast cancer. MATERIALS AND METHODS: The immunohistochemical expression of hTERT and c-Myc was investigated in 38 malignant breast tumours. The expression of hTERT was then correlated with the lymph node status, c-Myc expression and other clinicopathological parameters of the tumours. RESULTS: hTERT expression was positive in 27 (71%) of the 38 tumours. 15 (79%) of 19 node positive tumours were hTERT positive compared with 11 (63%) of 19 node negative tumours. The expression was higher in node positive tumours but this failed to reach statistical significance (p = 0.388). There was no significant association with tumour size, tumour grade or c-Myc expression. However, hTERT expression correlated positively with patients' age (correlation coefficient = 0.415, p = 0.0097). CONCLUSION: hTERT protein expression is independent of lymph node status, tumour size and grade and c-Myc protein expression in human breast cancer.
RESUMEN
INTRODUCTION: Deletion mapping studies have shown that genes in the region 3p21.3 are often deleted in epithelial malignancies. Although the regions deleted differ between individual cancers there appears to be a specificity for the type of malignancy produced. As a result this area of chromosome 3 is thought to contain multiple tumour suppressor genes. The present study concentrates on one gene in that region, APRG1. This gene codes for a protein, which has been implicated in cell membrane interactions. The aim was to determine using quantitative PCR whether the levels of this gene were negatively correlated with clinical outcome in breast cancer. METHODS: One hundred and twenty tumour tissues and 33 normal tissues were analyzed. Levels of transcription of APRG1 were determined using real-time quantitative PCR. APRG1 expression was normalized against CK19. Levels of expression were analyzed against staging, nodal involvement, grade, distant metastasis and survival over a 6 year follow up period. RESULTS: Levels of APRG1 mRNA were lower in malignant tissues. They fell further with increasing stage using the TNM classification This became statistically significant when TNM stages 3 and 4 were compared to TNM 1 (p = 0.0046, p = 0.04, t-test). They were lower in those with positive nodes although this did not reach statistical significance. There was a statistically significant reduction in APRG1 in grade 3 tumours cf. grade 1 (p = 0.0081). APRG1 expression was highly negatively correlated with progressive disease: alive with metastasis (p = 0.0069), local recurrence (p = 0.0055), died of breast cancer (p = 0.11), all progressive disease (p = 0.035). CONCLUSION: This study shows a compelling trend for APRG1 transcription levels to be lower in malignant tissues and lower still in those patients who develop progressive disease. There was also a statistically significant difference in APRG1 levels between grade 3 and grade 1 tumours. These results are highly suggestive of APRG1 acting as a tumour suppressor gene.
Asunto(s)
Neoplasias de la Mama/genética , Cromosomas Humanos Par 3/genética , Genes Supresores de Tumor , Neoplasias de la Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estadificación de Neoplasias , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
UNLABELLED: The Wnt family encodes secreted signaling molecules involved in cell adhesion and, by implication, cell growth. Wnt5a has been shown to behave as a putative oncogene and also as a tumour suppressor gene. This is a reflection of its role within a multi-step pathway and in the variety of ways in which its production can be stimulated or switched off. Wnt genes can be functionally separated into two classes; those that activate the canonical Wnt/beta-catenin pathway and those that activate the Wnt/Ca++ pathway. Wnt5a signals through frizzled receptors and, depending upon which frizzled receptor is present, may activate either pathway. Therefore the observed function of Wnt5a is entirely dependent upon its context, hence the confusion over its role in tumorigenesis. This study examines Wnt5a mRNA expression using RT-PCR in human breast cancer. MATERIALS AND METHODS: One hundred and twenty malignant breast tumours and 33 normal breast tissues were analysed. The levels of transcription of Wnt5a were determined using real-time quantitative PCR. Levels of expression were analysed against staging, nodal involvement, grade, distant metastasis and survival over a 6-year follow-up period. RESULTS: Levels of Wnt5a mRNA were lower in tumours than in normal tissue (mean values: 107 vs. 62.7). They fell further with increasing stage using the Nottingham Prognostic Index. This became statistically significant when NPI3 was compared to normal tissue (p=0.043, t-test). There was a trend towards lower levels of Wnt5a in those with progressive disease, however, this did not reach statistical significance. In patients with ER-negative disease, lower levels of Wnt5a were significantly associated with a worse clinical outcome (p=0.016). CONCLUSION: There is a trend for mRNA levels to be lower in cancerous tissue and lower still in those showing more aggressive behaviour. This is consistent with the hypothesis that Wnt5a is a tumour suppressor gene with potential clinical applications.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Neoplasias de la Mama/genética , Línea Celular Tumoral , Supervivencia sin Enfermedad , Humanos , Estadificación de Neoplasias , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Wnt , Proteína Wnt-5aRESUMEN
Attenuated retroviruses are currently the most widely used vectors in clinical gene therapy because of their potential to effect stable and permanent gene transfer. Since gene delivery is accompanied by random insertion of foreign genetic material into the recipient chromosomal DNA, the potential for insertional mutagenesis exists. In this study, we used a defective retrovirus vector containing a selectable marker, the hygromycin phosphotransferase gene, to investigate the mutagenic effects of vector integration on the mammalian genome. V79 Chinese hamster cells were infected with virus supernatants or by coculture with virus producer cells, and provirus insertion events occurred at low and high frequencies, respectively. The frequency of hprt mutagenesis was increased by a factor of 2.3 over the spontaneous hprt mutation frequency only following multiple provirus insertions/cell genome. Multiple provirus insertions (>3/genome) resulted in instability at the hprt locus in 63% of the virally induced hprt mutants, as indicated by rearrangements at the molecular level, whereas no rearrangements were found when the provirus copy number was 1-2/genome. To demonstrate direct proviral involvement in mutagenesis, the defective MLV vector was retrieved along with flanking genomic hprt sequences from one mutant, and localized within intron 5 of the hprt gene. These data suggest that provirus copy number is a key factor when considering the potential hazards of using retrovirus vectors for gene therapy.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/genética , Hipoxantina Fosforribosiltransferasa/genética , Mutagénesis Insercional/métodos , Provirus/genética , Retroviridae/genética , Línea Celular , Técnicas de Cocultivo , Reordenamiento Génico , Humanos , Masculino , Reacción en Cadena de la Polimerasa/métodosRESUMEN
AIMS: Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. hTERT (human telomerase reverse transcriptase) gene seems to be the rate-limiting determinant of telomerase reactivation. hTERT mRNA expression was reported to correlate with telomerase activity in cell lines and some human tumours. However the correlation between telomerase activity and hTERT mRNA expression has not been previously examined in human breast cancer. The present study aims to quantitatively measure the expression of hTERT mRNA and telomerase activity in human breast cancer and examine the relationship between these parameters. Furthermore the associations with other parameters including estrogen receptor (ER) and progesterone receptor (PgR) status, DNA ploidy, and S-phase fraction (SPF) are also examined. METHODS: RNA was extracted from 18 breast carcinomas and hTERT mRNA expressions were estimated by reverse transcriptase-PCR (RT-PCR) and Taqman methodology. These tumours had already been analysed for ER and PgR status using ligand-binding assays and had had their DNA ploidy and S-phase fractions measured by flow cytometry. Telomerase activity had already been determined by using a modified telomeric repeat and amplification protocol (TRAP) assay. RESULTS: The expression of hTERT mRNA in the breast tumours ranged between 1.3 and 2.7 x 10(7) copy numbers per micro g of cellular RNA (the median value was 2.7x10(5) and the mean was 3.1 x 10(6)). Telomerase activity was between 0 and 246 units of Total Protein Generated (TPG), where one unit of TPG was equal to 600 molecules, of telomerase substrate primers extended by at least three telomeric repeats. The median level of TPG was 60 units and the mean level was 81 units). Telomerase activity was found to significantly correlate with hTERT expression (r(s)=0.51112, P=0.0302). There was no significant correlation between hTERT and other parameters. CONCLUSION: hTERT mRNA expression significantly correlates with telomerase activity in human breast cancer. This is consistent with the hypothesis that hTERT is the catalytic and rate-limiting determinant subunit of the enzyme.
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Neoplasias de la Mama/enzimología , Telomerasa/análisis , Adulto , Anciano , Neoplasias de la Mama/genética , Proteínas de Unión al ADN , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Persona de Mediana Edad , Ploidias , ARN Mensajero/análisis , ARN Neoplásico/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fase S , Telomerasa/genéticaRESUMEN
Normal human keratinocytes possess a finite replicative lifespan. Most advanced squamous cell carcinomas (SCCs), however, are immortal, a phenotype that is associated with p53 and INK4A dysfunction, high levels of telomerase and loss of heterozygosity (LOH) at several genetic loci, suggestive of the dysfunction of other mortality genes. We show here that human chromosome 6 specifically reduces the proliferation or viability of a human SCC line, BICR31, possessing LOH across the chromosome. This was determined by an 88% reduction in colony yield (P<0.001), following the reintroduction of an intact normal chromosome 6 by monochromosome transfer. Deletion analysis of immortal segregants using polymorphic markers revealed the loss of a 2.9 Mbp interval, centred on marker D6S1045 at 6q14.3-q15, in 6/19 segregants. Crucially, allelic losses of this region were not identified in control hybrids constructed between chromosome 6 and the BICR6 SCC cell line that is heterozygous for chromosome 6 and which showed no reduction in colony formation relative to the control chromosome transfers. This indicates that the minimally deleted region at D6S1045 is not the result of fragile sites, a recombination hot spot, or a feature of the monochromosome transfer technique. LOH of D6S1045 was found in 2/9 immortal SCC lines and was part of a minimally deleted region of line BICR19. Furthermore, allelic imbalance, consistent with LOH, was detected in 3/17 advanced SCCs of the tongue. These results suggest the existence of a suppressor of SCC immortality and tumour development at chromosome 6q14.3-q15, which is important to a subset of human SCCs.
Asunto(s)
Carcinoma de Células Escamosas/genética , Cromosomas Humanos Par 6 , Eliminación de Gen , Carcinoma de Células Escamosas/mortalidad , Humanos , Pérdida de HeterocigocidadRESUMEN
Telomerase is a ribonucleoprotein enzyme that synthesises telomeres after cell division and maintains chromosomal length and stability thus leading to cellular immortalisation. hTERT (human telomerase reverse transcriptase) gene is the rate-limiting determinant of telomerase reactivation. The present study aims to quantitatively measure the expression of hTERT mRNA in human breast cancer, adjacent non-cancerous tissue (ANCT) and benign breast lesions, examine the association between hTERT and the clinicopathological characteristics of the cancer specimens and to explore the relationship between c-Myc and hTERT expressions. RNA was extracted from 49 breast carcinomas, 46 matched ANCT, and eight fibroadenomas. hTERT and c-Myc mRNA expressions were estimated by reverse transcriptase-PCR (RT-PCR) and Taqman methodology. hTERT mRNA was present in all of the cancerous and most of ANCT specimens with levels being much higher in the cancerous tissue than in ANCT. The ratio of hTERT mRNA in tumour to that in ANCT was 2011 (95% confidence interval 373-10,853, P < 0.0001). There was no significant association between tumour hTERT expression and patient's age, tumour size, grade, nodal metastasis, estrogen receptor (ER) positivity, lymphovascular (LVI) or c-Myc expression. However, there was a weak but significant negative correlation between hTERT expression and progesterone receptor (PR) status (p = 0.04) in tumours. hTERT mRNA expression was also significantly higher in carcinomas (median = 2.61 x 10(6)) than in fibroadenomas (median = 424).We conclude that hTERT mRNA expression is significantly higher in human breast cancer than in non-cancerous breast tissue suggesting that hTERT has a potential role in breast cancer diagnosis. The hTERT mRNA levels in tumour do not seem to be associated with the patient's age or advanced tumour stage. Furthermore, hTERT mRNA expression does not correlate with c-Myc mRNA expression in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Mama/metabolismo , Genes myc/genética , Telomerasa/genética , Mama/citología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Transformación Celular Neoplásica , Cartilla de ADN , Proteínas de Unión al ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estadificación de Neoplasias , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/metabolismoRESUMEN
Telomerase is required for the complete replication of chromosomal ends. In tumors, the human telomerase reverse transcriptase subunit (hTERT) is up-regulated, thereby removing a critical barrier for unlimited cell proliferation. To understand more about hTERT regulation, we measured hTERT RNA levels by quantitative reverse transcription (RT)-PCR. Telomerase-positive cell lines were found to contain between 0.2 and 6 molecules of spliced hTERT RNA per cell, whereas in telomerase-negative cells, the number of molecules was below the sensitivity of the assay (<0.004 molecules/cell). Intron-containing, immature hTERT RNA was observed only in nuclei of telomerase-positive cells, which suggests that hTERT RNA levels are transcriptionally regulated. Microcell transfer of a normal chromosome 3 into the human breast carcinoma cell line (21NT) abolishes telomerase activity and induces senescence. Endogenous hTERT transcripts were undetectable in the nuclei of 21NT-chromosome 3 hybrids, even in cells permanently expressing a transfected hTERT cDNA. However, chromosome 3 transfer did not affect the expression of green fluorescent protein reporter constructs driven by up to 7.4 kb of noncoding DNA flanking the 5' end of the hTERT gene. Because direct up-regulation of hTERT through c-Myc overexpression had previously been reported, we investigated whether chromosome 3 transfer affected c-Myc activity. An at least 30-fold reduction of immature intron-containing hTERT RNA was observed after the introduction of a normal chromosome 3, but expression levels of c-Myc, Mad1, and other c-Myc target genes were unchanged. Our results suggest that telomerase is regulated primarily at the level of hTERT transcription by complex mechanisms involving regulatory elements distant from the 5' flanking region, and that the putative hTERT repressor on chromosome 3 does not regulate the expression of hTERT through c-Myc or one of its coregulators.
Asunto(s)
Cromosomas Humanos Par 3/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas Proto-Oncogénicas c-myc/fisiología , ARN Mensajero/antagonistas & inhibidores , Telomerasa/antagonistas & inhibidores , Telomerasa/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Ciclo Celular/genética , Diferenciación Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Regulación hacia Abajo , Fibroblastos/enzimología , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Proto-Oncogénicas c-myc/genética , Empalme del ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Telomerasa/biosíntesis , Telomerasa/genética , Células Tumorales CultivadasRESUMEN
Acquisition of metastatic ability by prostate cancer cells is the hallmark of their lethal trait and outcome. However, the genetic alterations underlying the clinical progression and pathogenesis of prostate cancer are not well understood. Several studies involving loss of heterozygosity (LOH) and comparative genomic hybridization analysis have identified distinctively altered regions on various human chromosomes, and genomic imbalance of chromosome 20 was implicated in progression and recurrence of prostate tumors. To examine the role of chromosome 20 in prostate neoplasms, we introduced this chromosome into highly metastatic rat prostate cancer cells using the microcell-mediated chromosome transfer technique. Introduction of the chromosome resulted in significant suppression of the metastatic ability of the hybrid cells, by as much as 98%, without any interference with the in vivo growth rate or tumorigenicity of primary tumor in SCID mice. Our STS-PCR analysis on 10 hybrid clones indicates that the suppressor activity of chromosome 20 is located in the p11.23-12 region. Further examination of the hybrid clones by experimental metastasis assay and histologic analysis as well as Matrigel invasion assay suggests the involvement of the suppressor region at an early stage of invasion and extravasation. We also investigated the status of the chromosome 20 suppressor region in pathology specimens from human prostate cancer patients and detected the frequent loss of this region in high-grade tumors. These results suggest the presence of a putative suppressor gene on human chromosome 20 that is functionally involved in development of prostate cancer metastases.
Asunto(s)
Cromosomas Humanos Par 20/genética , Genes Supresores de Tumor/genética , Metástasis de la Neoplasia/genética , Anciano , Anciano de 80 o más Años , Animales , Mapeo Cromosómico , Perfilación de la Expresión Génica , Humanos , Células Híbridas/patología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia/patología , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Ratas , Lugares Marcados de Secuencia , Células Tumorales CultivadasRESUMEN
Human ribosomal gene repeats are distributed among five nucleolar organizer regions (NORs) on the p arms of acrocentric chromosomes. On exit from mitosis, nucleoli form around individual active NORs. As cells progress through the cycle, these mini-nucleoli fuse to form large nucleoli incorporating multiple NORs. It is generally assumed that nucleolar incorporation of individual NORs is dependent on ribosomal gene transcription. To test this assumption, we determined the nuclear location of individual human acrocentric chromosomes, and their associated NORs, in mouse> human cell hybrids. Human ribosomal genes are transcriptionally silent in this context. Combined immunofluorescence and in situ hybridization (immuno-FISH) on three-dimensional preserved nuclei showed that human acrocentric chromosomes associate with hybrid cell nucleoli. Analysis of purified nucleoli demonstrated that human and mouse NORs are equally likely to be within a hybrid cell nucleolus. This is supported further by the observation that murine upstream binding factor can associate with human NORs. Incorporation of silent NORs into mature nucleoli raises interesting issues concerning the maintenance of the activity status of individual NORs.