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1.
Front Oncol ; 14: 1406946, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39165691

RESUMEN

Introduction: Breast cancer (BC) is the most common cancer affecting women in the United States. Ductal carcinoma in situ (DCIS) is the earliest identifiable pre-invasive BC lesion. Estimates show that 14 to 50% of DCIS cases progress to invasive BC. Methods: Our objective was to identify nuclear matrix proteins (NMP) with specifically altered expression in DCIS and later stages of BC compared to non-diseased breast reduction mammoplasty and a contralateral breast explant culture using mass spectrometry and RNA sequencing to accurately identify aggressive DCIS. Results: Sixty NMPs were significantly differentially expressed between the DCIS and non-diseased breast epithelium in an isogenic contralateral pair of patient-derived extended explants. Ten of the sixty showed significant mRNA expression level differences that matched the protein expression. These 10 proteins were similarly expressed in non-diseased breast reduction cells. Three NMPs (RPL7A, RPL11, RPL31) were significantly upregulated in DCIS and all other BC stages compared to the matching contralateral breast culture and an unrelated non-diseased breast reduction culture. RNA sequencing analyses showed that these three genes were increasingly upregulated with BC progression. Finally, we identified three NMPs (AHNAK, CDC37 and DNAJB1) that were significantly downregulated in DCIS and all other BC stages compared to the isogenically matched contralateral culture and the non-diseased breast reduction culture using both proteomics and RNA sequencing techniques. Discussion: These genes should form the basis of, or contribute to, a molecular diagnostic panel that could identify DCIS lesions likely to be indolent and therefore not requiring aggressive treatment.

2.
bioRxiv ; 2024 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-38405693

RESUMEN

Breast cancer (BC) is the most common cancer affecting women in the United States. Ductal carcinoma in situ (DCIS) is the earliest identifiable pre-invasive BC lesion. Estimates show that 14 to 50% of DCIS cases progress to invasive BC. Our objective was to identify nuclear matrix proteins (NMP) with specifically altered expression in DCIS and later stages of BC compared to non-diseased breast reduction mammoplasty and a contralateral breast explant using mass spectrometry and RNA sequencing to accurately identify aggressive DCIS. Sixty NMPs were significantly differentially expressed between the DCIS and non-diseased breast epithelium in an isogenic contralateral pair of patient-derived extended explants. Ten of the sixty showed significant mRNA expression level differences that matched the protein expression. These 10 proteins were similarly expressed in non-diseased breast reduction cells. Three NMPs (RPL7A, RPL11, RPL31) were significantly upregulated in DCIS and all other BC stages compared to the matching contralateral breast culture and an unrelated non-diseased breast reduction culture. RNA sequencing analyses showed that these three genes were upregulated increasingly with BC progression. Finally, we identified three NMPs (AHNAK, CDC37 and DNAJB1) that were significantly downregulated in DCIS and all other BC stages compared to the isogenically matched contralateral culture and the non-diseased breast reduction culture using both proteomics and RNA sequencing techniques.

3.
Life Sci ; 281: 119746, 2021 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-34181965

RESUMEN

AIMS: Gulf War illness (GWI) is thought to be associated with exposures experienced by soldiers deployed in the 1991 Gulf War. A major question is how these exposures continue to influence the health of these individuals three decades later. One potentially permanent effect of such exposures is the induction of genetic mutations. We investigated whether veterans with GWI exhibited persistently elevated levels of somatic mutation. MATERIALS AND METHODS: We applied the blood-based glycophorin A (GPA) somatic mutation assay to a cohort of veterans diagnosed with GWI and a set of both concurrent and historic age-matched controls. This assay quantifies red blood cells with a phenotype consistent with loss of one allele at the genetic determinant for the MN blood group, the GPA gene. KEY FINDINGS: As a population, those affected with GWI exhibited an uninduced mutation frequency at the GPA locus that was effectively twice that observed in controls, a result that was statistically significant. This result was influenced by an increase in the incidence of individuals with aberrantly high mutation frequencies, seemingly higher than would be expected by dose extrapolation and consistent with the induction of localized genomic instability in the hematopoietic bone marrow stem cells. When these "outliers" were removed from consideration, the remaining affected population retained a significantly higher mean allele loss mutation frequency, suggesting that both dose-dependent bone marrow genotoxicity and induction of genomic instability are contributing to the elevation in mutation frequency in these affected veterans. SIGNIFICANCE: This study provides evidence that manifestation of GWI is associated with increased cumulative exposure to agents capable of inducing persistent mutations in bone marrow stem cells. Whether these mutations are involved in the clinical aspects of the condition or are simply biomarkers of overall exposure has yet to be determined. The increased incidence of genomic instability suggests that this persistent mutation can have important delayed effects on cellular integrity.


Asunto(s)
Inestabilidad Genómica , Mutación , Síndrome del Golfo Pérsico/genética , Veteranos , Estudios de Casos y Controles , Glicoforinas/genética , Humanos , Masculino
4.
Methods Mol Biol ; 2102: 349-359, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31989566

RESUMEN

The HPRT assay uses incorporation of toxic nucleotide analogues to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosphoribosyl transferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and the products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene.


Asunto(s)
Análisis Mutacional de ADN/métodos , Hipoxantina Fosforribosiltransferasa/genética , Dermatoglifia del ADN , ADN Complementario/biosíntesis , ADN Complementario/genética , Exones , Genes Reporteros , Humanos , Hipoxantina Fosforribosiltransferasa/metabolismo , Reacción en Cadena de la Polimerasa Multiplex , Mutación Puntual , Eliminación de Secuencia , Recombinación V(D)J , Flujo de Trabajo
5.
Mil Med ; 185(1-2): e47-e52, 2020 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-31334811

RESUMEN

INTRODUCTION: Veterans of the 1991 Gulf War were potentially exposed to a mixture of stress, chemicals and radiation that may have contributed to the persistent symptoms of Gulf War Illness (GWI). The genotoxic effects of some of these exposures are mediated by the DNA nucleotide excision repair (NER) pathway. We hypothesized that individuals with relatively low DNA repair capacity would suffer greater damage from cumulative genotoxic exposures, some of which would persist, causing ongoing problems. MATERIALS AND METHODS: Blood samples were obtained from symptomatic Gulf War veterans and age-matched controls. The unscheduled DNA synthesis assay, a functional measurement of NER capacity, was performed on cultured lymphocytes, and lymphocyte mRNA was extracted and analyzed by sequencing. RESULTS: Despite our hypothesis that GWI would be associated with DNA repair deficiency, NER capacity in lymphocytes from affected GWI veterans actually exhibited a significantly elevated level of DNA repair (p = 0.016). Both total gene expression and NER gene expression successfully differentiated individuals with GWI from unaffected controls. The observed functional increase in DNA repair capacity was accompanied by an overexpression of genes in the NER pathway, as determined by RNA sequencing analysis. CONCLUSION: We suggest that the observed elevations in DNA repair capacity and NER gene expression are indicative of a "hormetic," i.e., induced or adaptive protective response to battlefield exposures. Normally such effects are short-term, but in these individuals this response has resulted in a long-term metabolic shift that may also be responsible for the persistent symptoms of GWI.


Asunto(s)
Síndrome del Golfo Pérsico , Veteranos , ADN , Reparación del ADN , Femenino , Guerra del Golfo , Humanos , Masculino , Síndrome del Golfo Pérsico/genética
6.
BMC Med Genomics ; 11(1): 95, 2018 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-30376844

RESUMEN

BACKGROUND: Nucleotide Excision Repair (NER) is a major pathway of mammalian DNA repair that is associated with drug resistance and has not been well characterized in acute lymphoblastic leukemia (ALL). The objective of this study was to explore the role of NER in relapsed ALL patients. We hypothesized that increased expression of NER genes was associated with drug resistance and relapse in ALL. METHODS: We performed secondary data analysis on two sets of pediatric ALL patients that all ultimately relapsed, and who had matched diagnosis-relapse gene expression microarray data (GSE28460 and GSE18497). GSE28460 included 49 precursor-B-ALL patients, and GSE18497 included 27 precursor-B-ALL and 14 T-ALL patients. Microarray data were processed using the Plier 16 algorithm and the 20 canonical NER genes were extracted. Comparisons were made between time of diagnosis and relapse, and between early and late relapsing subgroups. The Chi-square test was used to evaluate whether NER gene expression was altered at the level of the entire pathway and individual gene expression was compared using t-tests. RESULTS: We found that gene expression of the NER pathway was significantly increased upon relapse in patients that took 3 years or greater to relapse (late relapsers, P = .007), whereas no such change was evident in patients that relapsed in less than 3 years (early relapsers, P = .180). Moreover, at diagnosis, the NER gene expression of the early relapsing subpopulation was already significantly elevated over that of the late relapsing group (P < .001). This pattern was validated by an 'NER score' established by averaging the relative expression of the 20 canonical NER genes. The NER score at diagnosis was found to be significantly associated with disease-free survival in precursor-B-ALL (P < .001). CONCLUSION: Patients are over two times more likely to undergo early relapse if they have a high NER score at diagnosis, hazard ratio 2.008, 95% CI (1.256-3.211). The NER score may provide a underlying mechanism for "time to remission", a known prognostic factor in ALL, and a rationale for differential treatment.


Asunto(s)
Reparación del ADN/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Algoritmos , Preescolar , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Lactante , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Recurrencia
7.
Neuromolecular Med ; 17(3): 297-304, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25989848

RESUMEN

Autism spectrum disorder is a heterogeneous disease, and numerous alterations of gene expression come into play to attempt to explain potential molecular and pathophysiological causes. Abnormalities of brain development and connectivity associated with alterations in cytoskeletal rearrangement, neuritogenesis and elongation of axons and dendrites might represent or contribute to the structural basis of autism pathology. Slit/Robo signaling regulates cytoskeletal remodeling related to axonal and dendritic branching. Components of its signaling pathway (ABL and Cdc42) are suspected to be molecular bases of alterations of normal development. The present review describes the most important mechanisms underlying neuritogenesis, axon pathfinding and the role of GTPases in neurite outgrowth, with special emphasis on alterations associated with autism spectrum disorders. On the basis of analysis of publicly available microarray data, potential biomarkers of autism are discussed.


Asunto(s)
Trastorno del Espectro Autista/etiología , Neuritas/patología , Neurogénesis , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/patología , Trastorno del Espectro Autista/fisiopatología , Transporte Axonal , Axones/fisiología , Biomarcadores , Encéfalo/patología , Conectoma , GTP Fosfohidrolasas/fisiología , Perfilación de la Expresión Génica , Conos de Crecimiento/fisiología , Humanos , Microtúbulos/fisiología , Modelos Neurológicos , Proteínas del Tejido Nervioso/fisiología , Plasticidad Neuronal , Proteínas Proto-Oncogénicas c-abl/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transducción de Señal , Proteína de Unión al GTP cdc42/fisiología
8.
Photochem Photobiol ; 91(2): 493-500, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25393451

RESUMEN

Nucleotide excision repair (NER) is an important modulator of disease, especially in constitutive deficiencies such as the cancer predisposition syndrome Xeroderma pigmentosum. We have found profound variation in NER capacity among normal individuals, between cell-types and during carcinogenesis. NER is a repair system for many types of DNA damage, and therefore many types of genotoxic carcinogenic exposures, including ultraviolet light, products of organic combustion, metals and oxidative stress. Because NER is intimately related to cellular metabolism, requiring components of both the DNA replicative and transcription machinery, it has a narrow range of functional viability. Thus, genes in the NER pathway are expressed at the low levels manifested by, for example, nuclear transcription factors. As NER activity and gene expression vary by cell-type, it is inherently epigenetically regulated. Furthermore, this epigenetic modulation is disregulated during sporadic breast carcinogenesis. Loss of NER is one basis of genomic instability, a required element in cellular transformation, and one that potentially influences response to therapy. In this study, we demonstrate differences in NER capacity in eight adult mouse tissues, and place this result into the context of our previous work on mouse extraembryonic tissues, normal human tissues and sporadic early stage human breast cancer.


Asunto(s)
Neoplasias de la Mama/genética , Transformación Celular Neoplásica/genética , Enzimas Reparadoras del ADN/genética , Reparación del ADN , ADN/genética , Epigénesis Genética/efectos de la radiación , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/etiología , Neoplasias de la Mama/patología , Carcinogénesis/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , ADN/metabolismo , Daño del ADN , Enzimas Reparadoras del ADN/metabolismo , Femenino , Variación Genética , Inestabilidad Genómica , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Especificidad de Órganos , Técnicas de Cultivo de Tejidos , Rayos Ultravioleta/efectos adversos
9.
Methods Mol Biol ; 1105: 193-202, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24623230

RESUMEN

This assay quantifies the extent of double-strand break (DSB) DNA damage in cell populations embedded in agarose and analyzed for migratory DNA using pulsed-field gel electrophoresis with ethidium bromide staining. The assay can measure preexisting damage as well as induction of DSB by chemical (e.g., bleomycin), physical (e.g., X-irradiation), or biological (e.g., restriction enzymes) agents. By incubating the cells under physiological conditions prior to processing, the cells can be allowed to repair DSB, primarily via the process of nonhomologous end joining. The amount of repair, corresponding to the repair capacity of the treated cells, is then quantified by determining the ratio of the fractions of activity released in the lanes in comparison to the total amount of DNA fragmentation following determination of an optimal exposure for maximum initial fragmentation. Repair kinetics can also be analyzed through a time-course regimen.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Electroforesis en Gel de Campo Pulsado , Animales , Células Cultivadas , ADN/genética , ADN/aislamiento & purificación , Fragmentación del ADN , Humanos , Mutágenos/toxicidad
10.
Methods Mol Biol ; 1105: 223-44, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24623232

RESUMEN

The glycophorin A assay concurrently detects and quantifies erythrocytes with allele-loss phenotypes at the autosomal locus responsible for the polymorphic MN blood group. It uses a pair of allele-specific monoclonal antibodies and flow cytometry to efficiently analyze a standard population of five million cells. Two distinct variant phenotypes are detected: simple allele loss and allele loss followed by reduplication of the remaining allele; both are consistent with the mechanisms underlying "loss of heterozygosity" at tumor-suppressor genes. The assay is an intermediate biomarker of biological effect in the somatic mutational model of human cancer and has been applied to populations with a known or suspected genotoxic exposure, to patients with hereditary syndromes causing predisposition to cancer (where the assay has been applied diagnostically), and to patients manifesting cancer as a disease endpoint.


Asunto(s)
Glicoforinas/genética , Alelos , Análisis Mutacional de ADN , Eritrocitos/fisiología , Citometría de Flujo , Eliminación de Gen , Humanos , Pérdida de Heterocigocidad , Fenotipo
11.
Methods Mol Biol ; 1105: 291-301, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24623237

RESUMEN

The HPRT assay uses incorporation of toxic nucleotide analogues to select for cells lacking the purine scavenger enzyme hypoxanthine-guanine phosphoribosyl transferase. A major advantage of this assay is the ability to isolate mutant cells and determine the molecular basis for their functional deficiency. Many types of analyses have been performed at this locus: the current protocol involves generation of a cDNA and multiplex PCR of each exon, including the intron/exon junctions, followed by direct sequencing of the products. This analysis detects point mutations, small deletions and insertions within the gene, mutations affecting RNA splicing, and products of illegitimate V(D)J recombination within the gene. Establishment of and comparisons with mutational spectra hold the promise of identifying exposures to mutation-inducing genotoxicants from their distinctive pattern of gene-specific DNA damage at this easily analyzed reporter gene.


Asunto(s)
Análisis Mutacional de ADN , Hipoxantina Fosforribosiltransferasa/genética , Secuencia de Bases , Células Cultivadas , Cartilla de ADN/genética , ADN Complementario/genética , Exones , Genes Reporteros , Humanos , Reacción en Cadena de la Polimerasa Multiplex , Mutación , Recombinación V(D)J
12.
EMBO Mol Med ; 5(2): 264-79, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23307470

RESUMEN

Wnt/ß-catenin signalling has been suggested to be active in basal-like breast cancer. However, in highly aggressive metastatic triple-negative breast cancers (TNBC) the role of ß-catenin and the underlying mechanism(s) for the aggressiveness of TNBC remain unknown. We illustrate that WNT10B induces transcriptionally active ß-catenin in human TNBC and predicts survival-outcome of patients with both TNBC and basal-like tumours. We provide evidence that transgenic murine Wnt10b-driven tumours are devoid of ERα, PR and HER2 expression and can model human TNBC. Importantly, HMGA2 is specifically expressed during early stages of embryonic mammogenesis and absent when WNT10B expression is lost, suggesting a developmentally conserved mode of action. Mechanistically, ChIP analysis uncovered that WNT10B activates canonical ß-catenin signalling leading to up-regulation of HMGA2. Treatment of mouse and human triple-negative tumour cells with two Wnt/ß-catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation. We demonstrate that WNT10B has epistatic activity on HMGA2, which is necessary and sufficient for proliferation of TNBC cells. Furthermore, HMGA2 expression predicts relapse-free-survival and metastasis in TNBC patients.


Asunto(s)
Neoplasias de la Mama/fisiopatología , Proliferación Celular , Receptor alfa de Estrógeno/deficiencia , Proteína HMGA2/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptor ErbB-2/deficiencia , Receptores de Progesterona/deficiencia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Receptor alfa de Estrógeno/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Proteína HMGA2/metabolismo , Humanos , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-2/genética , Receptores de Progesterona/genética , Regulación hacia Arriba , Proteínas Wnt/genética , Vía de Señalización Wnt , beta Catenina/genética
13.
Cancer Immunol Immunother ; 60(7): 919-29, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21409596

RESUMEN

Hydroxysteroid (17ß) dehydrogenase type 12 (HSD17B12) is a multifunctional isoenzyme functional in the conversion of estrone to estradiol (E2), and elongation of long-chain fatty acids, in particular the conversion of palmitic to archadonic (AA) acid, the precursor of sterols and the inflammatory mediator, prostaglandin E(2). Its overexpression together with that of COX-2 in breast carcinoma is associated with a poor prognosis. We have identified the HSD17B12(114-122) peptide (IYDKIKTGL) as a naturally presented HLA-A*0201 (HLA-A2)-restricted CD8(+) T-cell-defined epitope. The HSD17B12(114-122) peptide, however, is poorly immunogenic in its in vitro ability to induce peptide-specific CD8(+) T cells. Acting as an "optimized peptide", a peptide (TYDKIKTGL), which is identical to the HSD17B12(114-122) peptide except for threonine at residue 1, was required for inducing in vitro the expansion of CD8(+) T-cell effectors cross-reactive against the HSD17B12(114-122) peptide. In IFN-γ ELISPOT assays, these effector cells recognize HSD17B12(114-122) peptide-pulsed target cells, as well as HLA-A2(+) squamous cell carcinoma of the head and neck (SCCHN) and breast carcinoma cell lines overexpressing HSD17B12 and naturally presenting the epitope. Whereas growth inhibition of a breast carcinoma cell line induced by HSD17B12 knockdown was only reversed by AA, in a similar manner, the growth inhibition of the SCCHN PCI-13 cell line by HSD17B12 knockdown was reversed by E2 and AA. Our findings provide the basis for future studies aimed at developing cancer vaccines for targeting HSD17B12, which apparently can be functional in critical metabolic pathways involved in inflammation and cancer.


Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/metabolismo , Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias de Cabeza y Cuello/inmunología , Fragmentos de Péptidos/inmunología , 17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 17-Hidroxiesteroide Deshidrogenasas/genética , Adulto , Anciano , Mama/citología , Mama/enzimología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/patología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito T/inmunología , Femenino , Fibroblastos/citología , Fibroblastos/enzimología , Antígeno HLA-A2/inmunología , Neoplasias de Cabeza y Cuello/enzimología , Neoplasias de Cabeza y Cuello/patología , Humanos , Técnicas para Inmunoenzimas , Interferón gamma/metabolismo , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Citotóxicos/inmunología
14.
Proc Natl Acad Sci U S A ; 107(50): 21725-30, 2010 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-21118987

RESUMEN

The molecular etiology of breast cancer has proven to be remarkably complex. Most individual oncogenes are disregulated in only approximately 30% of breast tumors, indicating that either very few molecular alterations are common to the majority of breast cancers, or that they have not yet been identified. In striking contrast, we now show that 19 of 19 stage I breast tumors tested with the functional unscheduled DNA synthesis assay exhibited a significant deficiency of DNA nucleotide excision repair (NER) capacity relative to normal epithelial tissue from disease-free controls (n = 23). Loss of DNA repair capacity, including the complex, damage-comprehensive NER pathway, results in genomic instability, a hallmark of carcinogenesis. By microarray analysis, mRNA expression levels for 20 canonical NER genes were reduced in representative tumor samples versus normal. Significant reductions were observed in 19 of these genes analyzed by the more sensitive method of RNase protection. These results were confirmed at the protein level for five NER gene products. Taken together, these data suggest that NER deficiency may play an important role in the etiology of sporadic breast cancer, and that early-stage breast cancer may be intrinsically susceptible to genotoxic chemotherapeutic agents, such as cis-platinum, whose damage is remediated by NER. In addition, reduced NER capacity, or reduced expression of NER genes, could provide a basis for the development of biomarkers for the identification of tumorigenic breast epithelium.


Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Reparación del ADN , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/etiología , Daño del ADN , Femenino , Humanos , Análisis por Micromatrices , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo
15.
Stem Cells ; 28(6): 1008-18, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20506227

RESUMEN

Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC-like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell-specific promoter. As expected, GFP was expressed by the CSC-enriched populations. However, an unanticipated result was that these cells remained blocked in a CSC-like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC-like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immunocompromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC-specific chemotherapeutic agents, which can then be verified in primary CSCs.


Asunto(s)
Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula/métodos , Células Madre Neoplásicas/citología , Antineoplásicos/farmacología , Biomarcadores/análisis , Neoplasias de la Mama/química , Neoplasias de la Mama/genética , Diferenciación Celular , Línea Celular Tumoral , Humanos , Células Madre Neoplásicas/química , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Transcripción Genética
16.
Expert Rev Mol Med ; 11: e5, 2009 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-19193248

RESUMEN

Recent studies have suggested that the pineal hormone melatonin may protect against breast cancer, and the mechanisms underlying its actions are becoming clearer. Melatonin works through receptors and distinct second messenger pathways to reduce cellular proliferation and to induce cellular differentiation. In addition, independently of receptors melatonin can modulate oestrogen-dependent pathways and reduce free-radical formation, thus preventing mutation and cellular toxicity. The fact that melatonin works through a myriad of signalling cascades that are protective to cells makes this hormone a good candidate for use in the clinic for the prevention and/or treatment of cancer. This review summarises cellular mechanisms governing the action of melatonin and then considers the potential use of melatonin in breast cancer prevention and treatment, with an emphasis on improving clinical outcomes.


Asunto(s)
Antineoplásicos/uso terapéutico , Antioxidantes/metabolismo , Antioxidantes/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Melatonina/metabolismo , Melatonina/uso terapéutico , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/prevención & control , Ensayos Clínicos Controlados como Asunto , Femenino , Humanos , Neoplasias Mamarias Experimentales , Modelos Animales , Modelos Biológicos , Pronóstico , Transducción de Señal
17.
Cell Tissue Res ; 333(3): 461-7, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18575893

RESUMEN

DNA repair, a fundamental function of cellular metabolism, has long been presumed to be constitutive and equivalent in all cells. However, we have previously shown that normal levels of nucleotide excision repair (NER) can vary by 20-fold in a tissue-specific pattern. We have now successfully established primary cultures of normal ovarian tissue from seven women by using a novel culture system originally developed for breast epithelial cells. Epithelial cells in these cultures aggregated to form three-dimensional structures called "attached ovarian epispheres". The availability of these actively proliferating cell cultures allowed us to measure NER functionally and quantitatively by the unscheduled DNA synthesis (UDS) assay, a clinical test used to diagnose constitutive deficiencies in NER capacity. We determined that ovarian epithelial cells manifested an intermediate level of NER capacity in humans, viz., only 25% of that of foreskin fibroblasts, but still 2.5-fold higher than that of peripheral blood lymphocytes. This level of DNA repair capacity was indistinguishable from that of normal breast epithelial cells, suggesting that it might be characteristic of the epithelial cell type. Similar levels of NER activity were observed in cultures established from a disease-free known carrier of a BRCA1 truncation mutation, consistent with previous normal results shown in breast epithelium and blood lymphocytes. These results establish that at least three "normal" levels of such DNA repair occur in human tissues, and that NER capacity is epigenetically regulated during cell differentiation and development.


Asunto(s)
Reparación del ADN , Células Epiteliales/citología , Células Epiteliales/metabolismo , Glándulas Mamarias Humanas/citología , Ovario/citología , Adulto , Factores de Edad , Proteína BRCA1/genética , Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Células Cultivadas , Reparación del ADN/genética , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Heterocigoto , Humanos , Recién Nacido , Linfocitos/citología , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Mutación , Adulto Joven
18.
Urol Oncol ; 26(4): 378-85, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18367102

RESUMEN

(Z)-1-1-Dichloro-2,3-diphenylcyclopropane (A(II)) and (Z)-1,1-dichloro-2-(4-methoxyphenyl)-3-phenylcyclopropane [2-(4-methoxyphenyl)-A(II)] inhibit tubulin polymerization, PSA production, and the proliferation of human prostate cancer cells. The actions of the agents were studied in three transgenic adenocarcinomas of the mouse prostate (TRAMP) cell lines. Antiproliferative potencies were determined and cells treated with the more potent 2-(4-methoxyphenyl)-A(II) were examined for induction of apoptosis. Microarray analyses were conducted to determine the apoptosis-related genes up- and down-regulated by the agent. 2-(4-Methoxyphenyl)-A(II) concentration-dependently inhibited growth of all three cell lines. Fifty percent and 100% growth inhibitory and 50% lethal concentrations were determined to be 0.3, 1.5, and 5 muM, respectively. Minimum detectable apoptosis-inducing concentrations by ELISA were 0.10 to 0.14 muM. PARP cleavage and two-color flow cytometry assays verified apoptosis induction. Microarray analyses showed Bok and Siva-pending to be up-regulated and that Birc, Dad1, and Atf5 were down-regulated. 2-(4-methoxyphenyl)-A(II) inhibits proliferation and induces apoptosis in the in vivo-adaptable TRAMP cells, suggesting the compound should be further examined in preclinical models.


Asunto(s)
Anisoles/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Ciclopropanos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Neoplasias de la Próstata/patología , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología
19.
Pathol Oncol Res ; 13(4): 276-83, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-18158561

RESUMEN

Homozygous loss of activity at the breast cancerpredisposing genes BRCA1 and BRCA2 (FANCD1) confers increased susceptibility to DNA double strand breaks, but this genotype occurs only in the tumor itself, following loss of heterozygosity at one of these loci. Thus, if these genes play a role in tumor etiology as opposed to tumor progression, they must manifest a heterozygous phenotype at the cellular level. To investigate the potential consequences of somatic heterozygosity for a BRCA1 mutation demonstrably associated with breast carcinogenesis on background somatic mutational burden, we applied the two standard assays of in vivo human somatic mutation to blood samples from a manifesting carrier of the Q1200X mutation in BRCA1 whose tumor was uniquely ascertained through an MRI screening study. The patient had an allele-loss mutation frequency of 19.4 x 10(-6) at the autosomal GPA locus in erythrocytes and 17.1 x 10(-6) at the X-linked HPRT locus in lymphocytes. Both of these mutation frequencies are significantly higher than expected from age-matched disease-free controls (P < 0.05). Mutation at the HPRT locus was similarly elevated in lymphoblastoid cell lines established from three other BRCA1 mutation carriers with breast cancer. Our patient's GPA mutation frequency is below the level established for diagnosis of homozygous Fanconi anemia patients, but consistent with data from obligate heterozygotes. The increased HPRT mutation frequency is more reminiscent of data from patients with xeroderma pigmentosum, a disease characterized by UV sensitivity and deficiency in the nucleotide excision pathway of DNA repair. Therefore, this BRCA1-associated breast cancer patient manifests a unique phenotype of increased background mutagenesis that likely contributed to the development of her disease independent of loss of heterozygosity at the susceptibility locus.


Asunto(s)
Neoplasias de la Mama/genética , Genes BRCA1 , Heterocigoto , Mutación , Adulto , Femenino , Predisposición Genética a la Enfermedad , Glicoforinas/genética , Humanos , Hipoxantina Fosforribosiltransferasa/genética
20.
BMC Womens Health ; 6: 10, 2006 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-16800895

RESUMEN

BACKGROUND: Evidence-based screening guidelines are needed for women under 40 with a family history of breast cancer, a BRCA1 or BRCA2 mutation, or other risk factors. An accurate assessment of breast cancer risk is required to balance the benefits and risks of surveillance, yet published studies have used narrow risk assessment schemata for enrollment. Breast density limits the sensitivity of film-screen mammography but is not thought to pose a limitation to MRI, however the utility of MRI surveillance has not been specifically examined before in women with dense breasts. Also, all MRI surveillance studies yet reported have used high strength magnets that may not be practical for dedicated imaging in many breast centers. Medium strength 0.5 Tesla MRI may provide an alternative economic option for surveillance. METHODS: We conducted a prospective, nonrandomized pilot study of 30 women age 25-49 years with dense breasts evaluating the addition of 0.5 Tesla MRI to conventional screening. All participants had a high quantitative breast cancer risk, defined as > or = 3.5% over the next 5 years per the Gail or BRCAPRO models, and/or a known BRCA1 or BRCA2 germline mutation. RESULTS: The average age at enrollment was 41.4 years and the average 5-year risk was 4.8%. Twenty-two subjects had BIRADS category 1 or 2 breast MRIs (negative or probably benign), whereas no category 4 or 5 MRIs (possibly or probably malignant) were observed. Eight subjects had BIRADS 3 results, identifying lesions that were "probably benign", yet prompting further evaluation. One of these subjects was diagnosed with a stage T1aN0M0 invasive ductal carcinoma, and later determined to be a BRCA1 mutation carrier. CONCLUSION: Using medium-strength MRI we were able to detect 1 early breast tumor that was mammographically undetectable among 30 young high-risk women with dense breasts. These results support the concept that breast MRI can enhance surveillance for young high-risk women with dense breasts, and further suggest that a medium-strength instrument is sufficient for this application. For the first time, we demonstrate the use of quantitative breast cancer risk assessment via a combination of the Gail and BRCAPRO models for enrollment in a screening trial.

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