RESUMO
The principal feeding stimulants for the beetle Scolytus multistriatus Marsham from the twig bark of Ulmus americana L. have been identified as (+)-catechin-5-beta-D-xylopyranoside and lupeyl cerotate.
RESUMO
The Friend erythroleukemia virus complex contains no cell-derived oncogene. Transformation by this virus may therefore involve mutations affecting cellular gene expression. We provide evidence that inactivating mutations of the cellular p53 gene are a common feature in Friend virus-induced malignancy, consistent with an antioncogene role for p53 in this disease. We have shown that frequent rearrangements of the p53 gene cause loss of expression or synthesis of truncated proteins, whereas overexpression of p53 protein is seen in other Friend cell lines. We now demonstrate that p53 expression in the latter cells is also abnormal, as a result of missense mutations in regions encoding highly conserved amino acids. Three of these aberrant alleles obtained from cells from different mice were cloned and found to function as dominant oncogenes in gene transfer assays, supporting the view that certain naturally occurring missense mutations in p53 confer a dominant negative phenotype on the encoded protein.
Assuntos
Transformação Celular Neoplásica , Vírus da Leucemia Murina de Friend/genética , Genes Dominantes , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Fosfoproteínas/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Embrião de Mamíferos , Epitopos/análise , Amplificação de Genes , Leucemia Eritroblástica Aguda/microbiologia , Leucemia Experimental/microbiologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Proteínas Oncogênicas/análise , Proteínas Oncogênicas/imunologia , Fosfoproteínas/análise , Fosfoproteínas/imunologia , RNA/genética , RNA Antissenso , RNA Mensageiro/antagonistas & inibidores , Ratos , Ratos Endogâmicos F344 , Ribonucleases , Proteína Supressora de Tumor p53RESUMO
Rat embryo fibroblast clones transformed with the human papillomavirus type 16 E7 gene and the H-ras oncogene (ER clones) fall into two groups on the basis of endogenous p53 genotype, wild type or mutant. We have compared these clones with the aim of indentifying physiological differences that could be attributed to p53 protein function. We show that all ER clones, regardless of p53 gene status, are tumorigenic and metastatic in severe combined immunodeficiency mice. We demonstrate that only the wild-type p53 protein expressed in ER clones is functional on the basis of its site-specific double-stranded DNA-binding activity and its ability to confer a G1 delay on cells following treatment with ionizing radiation. These data indicate that disruption of the p53 growth-regulatory pathway is not a prerequisite for the malignant conversion of rat embryo fibroblasts expressing the E7 gene and mutant ras. Differences in phenotype that were correlated with loss of p53 protein function included the following: serum-independent growth of ER clones in culture, decreased tumor doubling time in vivo, and increased radioresistance. In addition, we demonstrate the p53-dependent G1 checkpoint alone does not determine radiosensitivity.
Assuntos
Transformação Celular Neoplásica , Fase G1/fisiologia , Genes p53 , Genes ras , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Animais , Sequência de Bases , Sítios de Ligação , Ciclo Celular/efeitos da radiação , Linhagem Celular Transformada , Sobrevivência Celular/efeitos da radiação , Células Clonais , Sequência Consenso , Embrião de Mamíferos , Fibroblastos , Citometria de Fluxo , Raios gama , Camundongos , Camundongos SCID , Dados de Sequência Molecular , Metástase Neoplásica/patologia , Transplante de Neoplasias , Oligodesoxirribonucleotídeos , Proteínas E7 de Papillomavirus , Ratos , Fatores de Transcrição/genética , Transplante Heterólogo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
A number of cellular and viral genes encode proteins that play a role in the establishment of normal cells in culture. In addition, these genes cooperate with activated ras genes to induce cellular transformation. We show that ras-dependent transformation of rat embryo fibroblasts is more efficient when two establishment genes are used together compared with one alone. Both quantitative and qualitative differences in the efficiency of transformation were detected. The number of transformed foci generated was greater than the sum of the foci obtained with ras and each of the establishment genes used separately. In addition, the foci had a distinct morphology. Synergism was seen between the HPV-16 E7 gene and certain mutant alleles of the cellular p53 gene as well as between E7 and c-myc.
Assuntos
Transformação Celular Neoplásica/genética , Fibroblastos/patologia , Genes Virais/genética , Genes myc/genética , Genes p53/genética , Papillomaviridae/genética , Alelos , Animais , Transformação Celular Neoplásica/patologia , Células Cultivadas , Embrião de Mamíferos/citologia , Genes Virais/fisiologia , Genes myc/fisiologia , Genes p53/fisiologia , Genes ras/genética , Genes ras/fisiologia , Teste de Complementação Genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos , Ratos Endogâmicos F344RESUMO
Rat embryo fibroblasts transformed with HPV-16 E7 and the Ha-ras oncogene (ER clones) fall into two distinct groups based on their endogenous p53 status, wild-type or mutant. We have taken advantage of such clones in order to study the p53 target genes by the differential display method of RNA fingerprinting. We have identified a cDNA clone, clone 16, that recognises a large transcript on Northern blots. The clone 16 transcript is overexpressed in ER cell lines that express wild-type p53 compared with ER cell lines that express mutant p53. Similar to the waf1/p21 gene, which is transcriptionally activated in cells treated with ionizing radiation in a p53-dependent manner, the clone 16 transcript was also induced in response to cell irradiation. The sequence of clone 16 exhibits a high homology to two members of RAL retroviral-like elements.
Assuntos
Genes p53/genética , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Células Cultivadas , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos , Rim/metabolismo , Camundongos , Dados de Sequência Molecular , Mutação , Papillomaviridae , RNA/metabolismo , Ratos , Fatores de Transcrição/metabolismo , Transfecção , Proteína Supressora de Tumor p53/genética , Proteínas WT1RESUMO
Heat shock protein 27 (Hsp27) is emerging as a promising therapeutic target for treatment of various cancers. Although the role of Hsp27 in protection from stress-induced intrinsic cell death has been relatively well studied, its role in Fas (death domain containing member of the tumor necrosis factor receptor superfamily)-induced apoptosis and cell proliferation remains underappreciated. Here, we show that Hsp27 silencing induces dual coordinated effects, resulting in inhibition of cell proliferation and sensitization of cells to Fas-induced apoptosis through regulation of PEA-15 (15-kDa phospho-enriched protein in astrocytes). We demonstrate that Hsp27 silencing suppresses proliferation by causing PEA-15 to bind and sequester extracellular signal-regulated kinase (ERK), resulting in reduced translocation of ERK to the nucleus. Concurrently, Hsp27 silencing promotes Fas-induced apoptosis by inducing PEA-15 to release Fas-associating protein with a novel death domain (FADD), thus allowing FADD to participate in death receptor signaling. Conversely, Hsp27 overexpression promotes cell proliferation and suppresses Fas-induced apoptosis. Furthermore, we show that Hsp27 regulation of PEA-15 activity occurs in an Akt-dependent manner. Significantly, Hsp27 silencing in a panel of phosphatase and tensin homolog on chromosome 10 (PTEN) wild-type or null cell lines, and in LNCaP cells that inducibly express PTEN, resulted in selective growth inhibition of PTEN-deficient cancer cells. These data identify a dual coordinated role of Hsp27 in cell proliferation and Fas-induced apoptosis via Akt and PEA-15, and indicate that improved clinical responses to Hsp27-targeted therapy may be achieved by stratifying patient populations based on tumor PTEN expression.
Assuntos
Apoptose , Proliferação de Células , Proteínas de Choque Térmico HSP27/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas/metabolismo , Receptor fas/metabolismo , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Proteínas de Choque Térmico HSP27/antagonistas & inibidores , Proteínas de Choque Térmico HSP27/genética , Humanos , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
AIMS/HYPOTHESIS: Insulin controls glucose metabolism via multiple signalling pathways, including the phosphatidylinositol 3-kinase (PI3K) pathway in muscle and adipose tissue. The protein/lipid phosphatase Pten (phosphatase and tensin homologue deleted on chromosome 10) attenuates PI3K signalling by dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate generated by PI3K. The current study was aimed at investigating the effect of haploinsufficiency for Pten on insulin-stimulated glucose uptake. MATERIALS AND METHODS: Insulin sensitivity in Pten heterozygous (Pten(+/-)) mice was investigated in i.p. insulin challenge and glucose tolerance tests. Glucose uptake was monitored in vitro in primary cultures of myocytes from Pten(+/-) mice, and in vivo by positron emission tomography. The phosphorylation status of protein kinase B (PKB/Akt), a downstream signalling protein in the PI3K pathway, and glycogen synthase kinase 3beta (GSK3beta), a substrate of PKB/Akt, was determined by western immunoblotting. RESULTS: Following i.p. insulin challenge, blood glucose levels in Pten(+/-) mice remained depressed for up to 120 min, whereas glucose levels in wild-type mice began to recover after approximately 30 min. After glucose challenge, blood glucose returned to normal about twice as rapidly in Pten(+/-) mice. Enhanced glucose uptake was observed both in Pten(+/-) myocytes and in skeletal muscle of Pten(+/-) mice by PET. PKB and GSK3beta phosphorylation was enhanced and prolonged in Pten(+/-) myocytes. CONCLUSIONS/INTERPRETATION: Pten is a key negative regulator of insulin-stimulated glucose uptake in vitro and in vivo. The partial reduction of Pten due to Pten haploinsufficiency is enough to elicit enhanced insulin sensitivity and glucose tolerance in Pten(+/-) mice.
Assuntos
Insulina/farmacologia , PTEN Fosfo-Hidrolase/genética , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Cruzamentos Genéticos , Desoxiglucose/metabolismo , Diabetes Mellitus Tipo 2/genética , Fluordesoxiglucose F18 , Triagem de Portadores Genéticos , Glucose/farmacologia , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Insulina/sangue , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Tomografia por Emissão de PósitronsRESUMO
Rat embryo fibroblasts transformed with the HPV-16 E7 gene and the activated c-H-ras gene fall into two distinct phenotypic classes. At high cell density, clones of one class form colonies in methylcellulose supplemented with low serum; at low cell density, these cells display responsiveness to mitogenic factors present in serum-free conditioned medium from rat embryo fibroblasts. In contrast, clones of the second class exhibit an absolute dependency on growth factors present in serum at all cell densities in the methylcellulose colony assay and fail to respond to conditioned medium. We find that the status of the endogenous p53 gene is tightly correlated with these two classes of clones. Clones of the first class contain missense mutations in the p53 gene and have lost the wild-type allele. Clones of the second class express wild-type p53 protein. The importance of mutant p53 expression in reducing the growth factor dependency of transformed clones was confirmed in a separate series of experiments in which rat embryo fibroblasts were transformed with three genes, E7 + ras + mutant p53. The growth behaviour of these triply transfected clones was similar to that of the E7 + ras clones expressing endogenous mutant p53. We demonstrate that the enhanced proliferation of E7 + ras clones expressing mutant p53 protein involves an autocrine mechanism.
Assuntos
Genes Virais , Genes p53 , Genes ras , Proteínas Oncogênicas Virais/biossíntese , Proteínas Oncogênicas/biossíntese , Papillomaviridae/genética , Animais , Sequência de Bases , Linhagem Celular Transformada , Células Clonais , Meios de Cultura Livres de Soro , Primers do DNA , Embrião de Mamíferos , Fibroblastos , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese , Proteínas Oncogênicas/genética , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Mutação Puntual , Reação em Cadeia da Polimerase , Ratos , Ratos Endogâmicos F344 , Mapeamento por Restrição , Transfecção , Proteína Supressora de Tumor p53/biossínteseRESUMO
Despite enormous progress in the understanding and treatment of disease during the 20th century, the amount of care individuals receive from health professionals is arguably less than in previous decades. Being in the presence of caring people who practised human caring has always been the bedrock of services to individuals who were ill. With the rise of scientific positivism in the mid-19th century, traditional ways of caring for sick people, not susceptible to scientific investigation and intervention, were either abandoned or discouraged. The spread of outcome-orientated health services has led to care being redefined as the provision of the finest form of treatment that is financially viable. The spectre of a service in which the human dimension of caring is either prescribed or seen as invalid gives cause for concern. This paper argues for urgent re-examination of what we understand by 'care'.
Assuntos
Assistência ao Paciente/história , Relações Profissional-Paciente , Cristianismo/história , Empatia , História do Século XVIII , História do Século XIX , História do Século XX , História Antiga , História Medieval , Hospitais/história , Humanos , Amor , Filosofia/história , Ocidente/históriaRESUMO
Based on its genomic sequence and its pathogenesis, murine gammaherpesvirus-68 (gammaHV-68) has been established as a tractable model for the study of viral infections caused by the human gammaherpesviruses, Epstein-Barr virus or human herpesvirus-8. Despite significant advances, the mechanisms responsible for gammaHV-68-induced alterations in the protective host response, and the accompanying virus-induced leukocytosis, are not clear. In the present study, we questioned whether viral infection resulted in endogenous interleukin-10 (IL-10) production that might alter the host response. Infection of C57BL/6 mice resulted in increased IL-10 expression, demonstrating that gammaHV-68 could induce endogenous production of this cytokine. Infected C57BL/6 mice demonstrated the characteristic splenomegaly associated with this viral infection, however, we were surprised to discover that the splenomegaly was greater in syngeneic mice genetically deficient in IL-10 (IL-10-/-). These results strongly suggested that endogenously produced IL-10 might serve to limit leukocytosis in wild-type mice. Quantification of viral burden demonstrated a significant elevation in C57BL/6 versus IL-10-/- mice, with increases in virus being observed in both the macrophage and B-lymphocyte populations. The decreased viral load in syngeneic IL-10-/- mice correlated with an increased expression of endogenous IL-12, suggesting a mechanism of protection that was IL-12 dependent. Taken together, these studies demonstrate a surprising dichotomy for endogenous IL-10 production during gammaHV-68 infection. While the lack of IL-10 results in increased IL-12 expression and a lower viral burden, IL-10-/- mice also experience an increased leukocytosis.
Assuntos
Gammaherpesvirinae , Infecções por Herpesviridae/imunologia , Interleucina-10/biossíntese , Leucocitose/virologia , Esplenomegalia/virologia , Animais , Linfócitos B/imunologia , Infecções por Herpesviridae/virologia , Interleucina-10/imunologia , Interleucina-12/biossíntese , Leucocitose/imunologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Esplenomegalia/imunologia , Carga ViralRESUMO
Murine gammaherpesvirus-68 (gammaHV-68) induces a lymphocytosis in mice and establishes a latent infection of B lymphocytes following intranasal administration in anaesthetized animals. Because gammaHV-68 is a gammaherpesvirus, it has been used as a model to understand the pathogenesis of Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8) infections. In this study, we investigated the unlikely possibility that gammaHV-68 could survive the harsh gastrointestinal environment to efficiently infect intestinal epithelial cells, and then disseminate from mucosal sites to cause systemic disease. Surprisingly, oral administration, or gastric instillation which by-passed the oral cavity, readily caused a systemic lymphocytosis and established a latent infection in splenic leukocytes. The finding that gammaHV-68 could readily infect adult mice following gastric instillation strongly suggested that intestinal epithelial cells could be productively infected. Unlike the more routinely used method of intranasal inoculation, gammaHV-68 given intragastrically resulted in lytic virus, viral RNA and viral DNA being present in isolated intestinal epithelial cells. Furthermore, gammaHV-68 RNA and DNA, but not latent virus, could be detected in epithelial cells as long as 30 days post-infection, suggesting that some of these cells might be persistently infected. Taken together, these studies demonstrate that gammaHV-68 can survive passage through the gastrointestinal tract and infect intestinal epithelial cells. Following infection of gut epithelial cells, gammaHV-68 can disseminate from mucosal sites to induce a systemic lymphocytosis which is similar to the disease induced following intranasal inoculation.
Assuntos
Gammaherpesvirinae/patogenicidade , Infecções por Herpesviridae/etiologia , Administração Oral , Animais , Sequência de Bases , Primers do DNA/genética , DNA Viral/genética , DNA Viral/isolamento & purificação , Células Epiteliais/virologia , Gammaherpesvirinae/genética , Gammaherpesvirinae/isolamento & purificação , Infecções por Herpesviridae/virologia , Humanos , Intestinos/virologia , Linfocitose/etiologia , Linfocitose/virologia , Camundongos , Camundongos Endogâmicos BALB C , RNA Viral/genética , RNA Viral/isolamento & purificação , Baço/virologia , Estômago , Fatores de TempoRESUMO
Stromal cell-derived factor-1 (SDF-1), a C-X-C family chemokine, is a potent T lymphocyte chemoattractant. We investigated the effects of T cell activation on the chemotactic response to SDF-1. Anti-CD3 Ab stimulation of either Jurkat T cells or murine peripheral CD4+ T lymphocytes produced a dramatic inhibition of SDF-1-induced chemotaxis. In contrast, the SDF-1 responses of Jurkat clones with deficiencies in key TCR signaling components (Lck, CD45, and TCR-beta), were only marginally reduced by anti-CD3 stimulation. Similar to PMA treatment, which abolished both CXCR4 receptor expression and the chemotactic response of Jurkat cells to SDF-1, anti-CD3 Ab treatment reduced cell surface expression of CXCR4 to 65% of the control value, an effect that was blocked by protein kinase C inhibitors. Our data suggest that initial T cell activation events inhibit the response of Jurkat T cells to CXCR4 stimulation. In contrast, SDF-1 treatment resulted in a reduction of tyrosine phosphorylation of the TCR downstream effectors, ZAP-70, SLP-76, and LAT (linker for activation of T cells), suggesting that this chemokine potentially regulates the threshold for T cell activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Quimiocinas CXC/antagonistas & inibidores , Quimiocinas CXC/fisiologia , Quimiotaxia de Leucócito/imunologia , Proteínas de Membrana , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores CXCR4/fisiologia , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Complexo CD3/imunologia , Proteínas de Transporte/metabolismo , Membrana Celular/imunologia , Membrana Celular/metabolismo , Quimiocina CXCL12 , Quimiotaxia de Leucócito/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Humanos , Células Jurkat/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Peso Molecular , Muromonab-CD3/farmacologia , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Fosforilação , Proteína Quinase C/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/biossíntese , Transdução de Sinais/imunologia , Células Estromais/fisiologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Proteína-Tirosina Quinase ZAP-70RESUMO
The roles of protein-tyrosine phosphatases (PTPs) in processes such as cell growth and adhesion are poorly understood. To explore the ability of specific PTPs to regulate cell signaling pathways initiated by stimulation of growth factor receptors, we expressed the receptor-like PTP, PTPalpha, in A431 epidermoid carcinoma cells. These cells express high levels of the epidermal growth factor (EGF) receptor and proliferate in response to the autocrine production of transforming growth factor-alpha. Conversely, EGF stimulation of A431 cells in vitro leads to growth inhibition and triggers the rapid detachment of these cells from the substratum. Although PTPalpha expression did not alter the growth characteristics of either unstimulated or EGF-stimulated cells, this phosphatase was associated with increased cell-substratum adhesion. Furthermore, PTPalpha-expressing A431 cells were strikingly resistant to EGF-induced cell rounding. Overexpression of PTPalpha in A431 cells was associated with the dephosphorylation/activation of specific Src family kinases, suggesting a potential mechanism for the observed alteration in A431 cell-substratum adhesion. Src kinase activation was dependent on the D1 catalytic subunit of PTPalpha, and there was evidence of association between PTPalpha and Src kinase(s). PTPalpha expression also led to increased association of Src kinase with the integrin-associated focal adhesion kinase, pp125(FAK). In addition, paxillin, a Src and/or pp125(FAK) substrate, displayed increased levels of tyrosine phosphorylation in PTPalpha-expressing cells and was associated with elevated amounts of Csk. In view of these alterations in focal adhesion-associated molecules in PTPalpha-expressing A431 cells, as well as the changes in adhesion demonstrated by these cells, we propose that PTPalpha may have a role in regulating cell-substratum adhesion.
Assuntos
Adesão Celular/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Carcinoma de Células Escamosas , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Clonagem de Organismos , Proteínas do Citoesqueleto/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fator de Crescimento Epidérmico/fisiologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Cinética , Dados de Sequência Molecular , Paxilina , Fragmentos de Peptídeos/química , Fosfopeptídeos/química , Fosfoproteínas/metabolismo , Proteínas Tirosina Fosfatases/biossíntese , Receptor de Insulina/metabolismo , Proteínas Recombinantes/metabolismo , Corantes de Rosanilina/metabolismo , Especificidade por Substrato , Transfecção , Fator de Crescimento Transformador alfa/farmacologia , Fator de Crescimento Transformador alfa/fisiologia , Células Tumorais CultivadasRESUMO
A point mutation in the mRNA of NADP(H): quinone oxidoreductase 1 (NQO1, DT-diaphorase) is believed to be responsible for reduced enzyme activity in the adenocarcinoma BE cell line. The present study examined nine cultured human non-cancerous fibroblast cell strains, five of which were from members of a single cancer-prone family, which demonstrated widely varying activity levels of DT-diaphorase (41 - 3462 nmol min-1 mg-1 protein), to determine if genetic alteration of the NQO1 or NOQ2 gene was involved in determining enzyme activity. All cell strains expressed NQO1 and NQO2 mRNA as measured by a quantitative polymerase chain reaction amplification technique. No relationship was found between the level of mRNA expressed and the enzyme activity in the cells. Sequencing of the entire complementary DNA from the cell strains revealed only a single base substitution at nucleotide 609 in one allele encoding NQO1 in every cell strain from members of the cancer-prone family, except for one cell strain which expressed only the T at nucleotide 609 in both alleles. Subsequent examination of genomic DNA from 44 individuals revealed that this base substitution is present in approximately 50% of the population. The presence of the T at nucleotide 609 in the NQO1 locus does not appear to be directly causal for altered DT-diaphorase activity.