Report of the clinical practice task force survey of the clinical cytometry society.
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Antibodies, Monoclonal↗
Biomedical subjects
B H Davis
Publications and source records attributed to B H Davis.
Optimal number of reagents required to evaluate hematolymphoid neoplasias: results of an international consensus meeting.
At the ISAC 2000 Congress, the Clinical Cytometry Society organized a meeting of international experts to reach consensus on the minimum number of antibodies required for a full evaluation of hematologic and lymphoid neoplasias. A questionnaire was distributed prior to the meeting to numerous experts from US and European institutions and 13 responses were received. At the meeting, 25 individuals, including most of those who returned responses, participated in the discussions and voted on the issues presented. In chronic lymphoproliferative disorders (CLD), 9 antibodies (anti-CD5, CD19, kappa, lambda, CD3, CD20, CD23, CD10, and CD45) were deemed essential for initial evaluation by 75% of the participants. There was near unanimity that additional markers (selected from CD22, FMC7, CD11c, CD103, CD38, CD25, CD79b and heavy chains for B-cell disorders, and CD4, CD7, CD8, CD2, CD56, CD16, TCRa/b, and TCRg/d for T-cell disorders) would be needed to fully characterize CLD, although not every marker would be useful in all cases. Tissue lymphomas were believed to be similar to CLD, needing a minimum of 12--16 markers. However, for some cases, CD30, bcl-2, TdT, CD71, CD1a, and CD34 were cited as useful by the participants. Markers mentioned for plasma cell disorders included kappa, lambda, CD38, CD45, CD56, CD19, CD20, CD138, and heavy chains. Of 17 voting participants, 16 agreed that between 5 to 8 markers would be essential reagents for plasma cell disorders. For acute leukemia (AL), 10 markers (CD10, CD19, CD13, CD33, CD34, CD45, CD7, CD14, CD3, and HLADR) were considered essential by 75% of participants for initial characterization of the leukemia lineage. Most (>75%) agreed that at least one more B (CD20, CD22, CD79a, IgM), T (CD1a, CD2, CD4, CD5, CD8), myeloid (CD11b, CD15, CD64, CD117, myeloperoxidase), erythroid (CD36, CD71, glycophorin A), and megakaryocytic (CD41, CD61) reagents should be included in the essential panel. However, there was no agreement as to which was optimal. Thus, approximately 13--15 of those reagents would be considered essential in all cases of AL, whereas others (CD16, CD56, CDw65, TdT, and cytoplasmic CD3) were mentioned as useful in some cases. Almost all voting participants believed that the appropriate number of markers for complete characterization of AL would average 20--24. The majority of the responders (11 of 13) indicated that fewer reagents could be used in monitoring or staging patients with previously characterized disease, but not all ventured a specific number of reagents. From the above results, we conclude that the phenotypic analysis of hematologic and lymphoid neoplasia requires a rather extensive panel of reagents. Supplementary reagents might even be necessary if they prove to become relevant for diagnostic purposes. Reducing the number of antibodies could significantly compromise the diagnostic accuracy, appropriate monitoring, or therapy of these disorders.
Biomarkers↗
Diagnostic advances in defining erythropoietic abnormalities and red blood cell diseases.
The majority of clinical applications of flow cytometry begin with various approaches to remove red blood cells (RBCs) from the clinical sample. However, multiparameter cytometry has and will continue to contribute much to the understanding of the pathophysiology and diagnostic accuracy in the clinical evaluation of human diseases affecting erythroid cells. This review summarizes the diagnostic advances relating to erythroid cells in the areas of immunohematology, laboratory hematology, and infectious disease with particular emphasis on medical evaluation of the anemic patient and fetomaternal hemorrhage. Semin Hematol 38:148-159.
Animals↗
Increased distribution and expression of CD64 on blood polymorphonuclear cells from patients with the systemic inflammatory response syndrome (SIRS).
Evidence is growing to suggest that the multiple organ damage of the systemic inflammatory response syndrome (SIRS) arises from the untoward activity of blood polymorphonuclear cells (PMNs), which upon activation acquire the IgG high affinity receptor, CD64. In the current study, flow cytometry was used to assess the prevalence of CD64-bearing PMNs and the intensity of expression of CD64 in whole blood samples from 32 SIRS patients, 11 healthy normal subjects and from eight non-SIRS patients in the intensive care unit (ICU). The percentage of PMNs expressing CD64 was higher in SIRS patients (mean 65%) than in non-SIRS patients (mean 42%; P < 0.02) and in healthy controls (mean 19%; P < 0.001) and was particularly evident in patients with SIRS and sepsis (mean 71%; P < 0.02) as opposed to SIRS alone (mean 55%). There were more CD64 molecules expressed on PMNs from patients with SIRS (median 1331 molecules/cell) in comparison with PMNs from healthy subjects (median 678 molecules/cell; P < 0.01). The highest intensity of CD64 expression was associated with PMNs from patients with both SIRS and sepsis. Functional studies revealed that the supranormal binding of PMNs from patients with SIRS to endothelial monolayers treated with TNFalpha was impeded by anti-CD64 antibodies (mean 24% inhibition; P < 0.01). Monitoring the distribution of CD64+ PMNs and their level of CD64 expression could be of assistance in the rapid discrimination of patients with SIRS from other ICU patients and in the identification of PMNs which are likely to participate in the pathological manifestations of the disease.
Adult↗
Flow cytometric method for simultaneous assay of foetal haemoglobin containing red cells, reticulocytes and foetal haemoglobin containing reticulocytes.
Level of foetal haemoglobin (HbF) containing red cells (F cells) is a parameter for monitoring sickle cell anaemia (SS) patients undergoing treatment with HbF modulating drugs (including hydroxyurea (HU)). One convenient technique for F cell assay is flow cytometry. A flow cytometric method for the simultaneous assay of F cells, reticulocytes and HbF-containing reticulocytes (F reticulocytes) is described in this paper. These three parameters can be obtained within 2 h using double colour staining flow cytometry. Glutaraldehyde fixation, Triton X-100 permeabilization, monoclonal antibody to HbF Tri-colour conjugate (MoAb-HbF-TC; deep-red fluorescence) immuno-staining and thiazole orange (TO; green fluorescence) are employed. The red cell gate was set on forward scatter (FSC) and logarithmic side scatter (logSSC) for 50 000 cells on the flow cytometer. Fluorescent signals were acquired from fluorescent channel 1 (FL1; green) and (FL4; deep-red). Coefficient of variation percent (%CVs) of intra- and inter-assay were less than 9% and 15%, respectively. EDTA, citrate, heparin and CTAD anticoagulants are all suitable; the samples can be stored at 4 degrees C for up to 6 days. The method is a simple, rapid, convenient, reproducible and useful way of determining F cell, reticulocyte and F reticulocyte levels in sickle cell and thalassaemic patients.
Anemia, Sickle Cell↗
Hepatosplenic gamma/delta T-cell lymphoma in immunocompromised patients. Report of two cases and review of literature.
We describe 2 male patients in whom hepatosplenic gamma/delta T-cell lymphoma (HSTL) developed 6 and 10 years after renal transplantation. The onset was abrupt with systemic symptoms, cytopenia, and hepatosplenomegaly. The histologic examination of the spleen (case 1), liver, and bone marrow revealed sinusoidal infiltrates of markedly abnormal lymphocytes. The neoplastic cells in these cases were CD2+, CD3+, CD4-, CD5-, CD7+, CD8+, CD16+, CD56+, beta F1-negative, and TIA-1-negative. Both cases displayed clonal rearrangement of the T-cell receptor (TCR) delta gene and the TCR beta gene. The spleen in case 1 was positive for Epstein-Barr virus genome and showed TCR-gamma gene rearrangement by polymerase chain reaction. Isochromosome 7 [i(7)(q10)] was found in each case. Both patients died within 4 months of diagnosis. HSTL has been reported in only 5 renal transplant recipients. HSTL may be relatively more frequent in immunocompromised patients compared with the general population.
Adult↗
Diagnostic usefulness of CD23 and FMC-7 antigen expression patterns in B-cell lymphoma classification.
CD23 and FMC-7 are normal B-cell antigens used during diagnostic immunophenotyping of suspected lymphoproliferative disorders, but the diagnostic usefulness of antigenic expression patterns of simultaneous 2-color staining and flow cytometric analysis has not been reported. We evaluated the FMC-7 and CD23 expression pattern in 201 cases of B-cell lymphoma from tissue biopsy specimens by multiparameter flow cytometry. The CD23-/FMC-7+ pattern was the most common pattern in large cell, mantle cell, and marginal zone lymphomas. The CD23 and FMC-7 antigen, along with the CD5 coexpression pattern, permitted accurate classification of all 71 cases of small lymphocytic, mantle cell, and marginal zone types of lymphoma. The widest variation of patterns was with follicular cell lymphoma, although most cases expressed the CD23 +/-/FMC-7+ pattern (+/-, partial or minor subset expression). The CD23 and FMC-7 antigen expression pattern was predictive of subtypes in more than 95% of lymphoma cases and could narrow the differential diagnosis in the remaining cases. We conclude the flow cytometric CD23/FMC-7 expression pattern achieved by dual staining facilitates accurate and reproducible classification of B-cell lymphomas and has diagnostic usefulness.
Algorithms↗
Diagnostic utility of red cell flow cytometric analysis.
Although most of the diagnostic applications of flow cytometry bring forth examples of leukocyte immunophenotyping for immunodeficiency diseases and leukemia-lymphoma diagnosis, the same technology has improved medical assessment of diseases affecting the red cell and erythropoiesis. Flow cytometric methods were first applied to laboratory hematology with the improvement in reticulocyte counting and the creation of the immature reticulocyte fraction for better anemia evaluation and therapeutic monitoring. A more recent improvement attributable to flow cytometry is accurate detection of fetal red cells in the evaluation of FMH hemorrhage. The same method used in the detection of fetal RBCs based on HbF content measurement using monoclonal antibodies also offers the potential for enumeration of F cells, which promises to have use in therapeutic monitoring of patients with sickle cell disease and the evaluation of other hemoglobinopathies and myelodysplasia. Other clinical uses of flow cytometric RBC analysis include nonisotopic red cell survival studies, sensitive blood group typing, sensitive detection of immune-mediated hemolytic diseases, and evaluation of parasitic diseases whose life cycle involves intracellular RBC infestation. This article summarizes red cell flow cytometry, particularly as it impacts the areas of immunohematology and laboratory hematology, and points to areas of potential future contribution of this technology to diagnostic medicine.
Adult↗
Simplified flow cytometric method for fetal hemoglobin containing red blood cells.
Assay of fetal hemoglobin (HbF) and/or HbF containing red blood cells (F+ cells) is essential for monitoring sickle cell and thalassemic patients, especially during treatment with HbF stimulators. Some previous flow cytometric methods contain several washing steps. This simplified method contains no washing step and takes less than an hour to perform. The %F+ cells in five mixtures of fetal red blood cells with adult red blood cells were nonsignificantly different in the original and simplified procedure. The %F+ cells of 12 patients compared in these two procedures were also not significantly different. The intra- and interassay %CVs do not exceed 3% and 7% respectively. EDTA, citrate, or heparin is suitable as anticoagulant and the samples can be stored at 4 degrees C for up to 2 weeks. The %F+ cells and %HbF [by high-performance liquid chromatography (HPLC)] of 83 samples were highly significantly correlated regardless of diagnosis. In conclusion, this new simplified flow cytometric method for F+ cells is simple, convenient, rapid, reproducible, and could be applied for monitoring sickle cell and thalassemic patients as an alternative to HPLC, where this is unavailable. It can also be applied as a fetal cell assay in fetomaternal hemorrhage.
Adult↗
Proposed flow cytometric reference method for the determination of erythroid F-cell counts.
BACKGROUND: Quantitation of adult erythrocytes (RBC) containing fetal hemoglobin (F cells) is of potential clinical utility in evaluating erythropoietic disorders, such as myelodysplasia and hemoglobinopathies, and in monitoring F-cell augmenting therapy. F-cell counting methodologies include fluorescence microscopy and flow cytometry. Previous flow cytometric methods have employed an isotype antibody control to distinguish F cells from non-F cells. We investigated the feasibility of using the orange autofluorescence signal (FL2) in glutaraldehyde-fixed RBC to substitute for fluorescein isothiocyanate (FITC)-labeled isotype control antibody use in F-cell quantitation. METHODS: Our previously published method for fetal red cell detection in fetomaternal hemorrhage was used, employing a FITC-labeled anti-hemoglobin F (HbF) monoclonal antibody reagent. Blood samples with varying F-cell counts were quantitated for F cells using both immunofluorescence microscopy and flow cytometry comparing FITC-labeled isotype to FL1 thresholding defined by FL2 autofluorescence. RESULTS: F cell percentages obtained by using an FL2 defined threshold for FL1 gating correlated well with expected values in diluted blood samples (r(2) = 0.994, slope = 1. 019, intercept = 0.24), values obtained using an isotype control (r(2) = 0.996, slope = 1.012, intercept = -0.17), and microscopic immunofluorescence counts (r(2) = 0.989, slope = 0.999, intercept = -0.72). F-cell quantitation by the isotype control and FL2 autofluorescence methods was also comparable in 40 blood samples (r(2) = 0.994, slope = 1.014, intercept = 0.03). Intra-assay, interobserver, and interinstrument precision with this autofluorescence gating method exhibited low imprecision (coefficient of variation <14%). CONCLUSION: This novel method is a more objective and less laborious alternative for F-cell quantitation by flow cytometry compared to using an isotype control or microscopy, thereby providing a more robust methodology for clinical studies and consideration as a laboratory reference method for F-cell counting.
Adult↗
Platelet-derived growth factor is a principal inductive factormodulating mannose 6-phosphate/insulin-like growth factor-II receptorgene expression via a distal E-box in activated hepatic stellate cells.
Hepatic stellate cells (HSCs) become activated during the earlystages of hepatic injury associated with fibrogenesis. The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGFIIR) plays animportant role in early fibrogenesis by participating in the activationof latent transforming growth factor-beta, a potent inducer of thematrix proteins in activated stellate cells that produce the hepaticnodule. Platelet-derived growth factor (PDGF), a potent HSC mitogen, isreleased early in hepatic injury and activates several signallingpathways in HSCs. In this study we examined the role of PDGF-BB in HSCregulation of M6P/IGFIIR gene expression. Several promoter elementswere found and characterized that modulate M6P/IGFIIR expression inactivated stellate cells. The presence of a distal CACGTG E-box at-2695 was required for M6P/IGFIIR expression in transfectedstellate cells. When the distal E-box was removed there was no significant M6P/IGFIIR promoter activity. The distal E-box-binding protein responded specifically to PDGF-BB with increased binding. This coincided with PDGF-BB up-regulation of M6P/IGFIIR mRNA transcript levels. Downstream elements include two proximal (-2 to-48) CACGTG E-boxes that bind a different protein to the distal(-2695) E-box. The proximal E-boxes respond moderately to PDGF-BB. The promoter segment encompassing -144 to +109 is able to respond dramatically to serum but is refractory to PDGF-BB. However, a constitutively bound protein binding to the -611/-716 fragment appears to be a repressor that suppresses inductive changes in protein binding occurring downstream of -611. These results indicate that the M6P/IGFIIR promoter responds primarily and specifically to PDGF-BB through a distal E-box element and possibly through two proximal E-box elements.
Animals↗
Myelodysplastic syndrome at a large tertiary care community hospital: analysis according to the international prognostic scoring system.
The outcome of patients diagnosed myelodysplastic syndromes (MDS) between 1990 and 1997 from William Beaumont Hospital (WBH) was analyzed according to the International Prognostic Scoring System (IPSS) risk categorization. A retrospective study of 195 MDS patients wa s performed. Seventy-nine patients with MDS, in whom a karyotype was obtained and with an adequate follow-up were included in the final analysis. Cases of proliferative CMML (WBC > 12x10(9)/l) were excluded from the study. The overall median survival was 3.1 years, and median survival stratified by IPSS was 3.4, 4.1 and 0.5 years for the INT-1, INT-2 and high risk group and not yet reached for the low risk group. The overall survival by IPSS subcategorization were 6.88, 5.29, 5.30 and 2.12 years for the low, INT-1, INT-2, and high risk groups respectively. Cytogenetics were significant in predicting the overall survival. The IPSS score stratified patients into risk categories for development of AML. The risk of development into AML was 8, 8, 33 and 54% for the low, INT-1, INT-2 and high risk groups, respectively. We conclude that IPSS score can be useful in predicting survival and AML evolution in some MDS patients.
Acute Disease↗
The DNA binding protein BTEB mediates acetaldehyde-induced, jun N-terminal kinase-dependent alphaI(I) collagen gene expression in rat hepatic stellate cells.
Alcohol-induced cirrhosis results partially from the excessive production of collagen matrix proteins, which, predominantly alphaI(I) collagen, are produced and secreted by activated hepatic stellate cells (HSC). The accumulation of alphaI(I) collagen in HSC during cirrhosis is largely due to an increase in alphaI(I) collagen gene expression. Acetaldehyde, the major active metabolite of alcohol, is known to stimulate alphaI(I) collagen production in HSC. However, the mechanisms responsible for it remain unknown. The aim of this study was to elucidate the mechanisms by which alphaI(I) collagen gene expression is induced by acetaldehyde in rat HSC. In the present study, the acetaldehyde response element was located in a distal GC box, previously described as the UV response element, in the promoter of the alphaI(I) collagen gene (-1484 to -1476). The GC box was predominantly bound by the DNA binding transcription factor BTEB (basic transcription element binding protein), expression of which was acetaldehyde and UV inducible. Blocking BTEB protein expression significantly reduced the steady-state levels of the acetaldehyde-induced alphaI(I) collagen mRNA, suggesting that BTEB is required for this gene expression. Further studies found that acetaldehyde activated Jun N-terminal kinase (JNK) 1 and 2 and activator protein 1 (AP-1) transactivating activity. Inhibition of JNK activation resulted in the reduction of the acetaldehyde-induced BTEB protein abundance and alphaI(I) collagen mRNA levels, indicating that the expression of both genes is JNK dependent in HSC. Taken together, these studies demonstrate that BTEB mediates acetaldehyde-induced, JNK-dependent alphaI(I) collagen gene expression in HSC.
Acetaldehyde↗
1,25-Dihydroxyvitamin D(3) stimulates activator protein-1-dependent Caco-2 cell differentiation.
1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is a potential chemopreventive agent for human colon cancer. We have reported that 1,25(OH)(2)D(3) specifically activated protein kinase C-alpha (PKC-alpha) and also caused a reduction in proliferation while increasing apoptosis and differentiation in CaCo-2 cells, a cell line derived from a human colon cancer. The mechanisms by which this secosteroid influences these important cellular processes, however, remain unclear. The transcription factor, activator protein-1 (AP-1), regulates many genes involved in these processes. Therefore, we asked whether 1,25(OH)(2)D(3) activated AP-1 in CaCo-2 cells and, if so, by what mechanisms? 1,25(OH)(2)D(3) caused a time-dependent increase in AP-1 DNA binding activity and significantly enhanced the protein and mRNA abundance of c-Jun, a component of AP-1. 1, 25(OH)(2)D(3) also induced a rapid and transient activation of ERK2 (where ERK is extracellular signal-regulated kinase) and a more persistent activation of JNK1 (where JNK Jun N-terminal kinase). Transfection experiments revealed that 1,25(OH)(2)D(3) also increased AP-1 gene-transactivating activity. This AP-1 activation was completely blocked by PD 098059, a specific mitogen-activated protein kinase/ERK kinase inhibitor, as well as by a dominant negative JNK or a dominant negative Jun, indicating that the AP-1 activation induced by 1,25(OH)(2)D(3) was mediated by ERK and JNK. Using a specific inhibitor of the Ca(2+)-dependent PKC isoforms, Gö6976, and CaCo-2 cells stably transfected with antisense PKC-alpha cDNA, demonstrated that PKC-alpha mediated the AP-1 activation induced by this secosteroid. Inhibition of JNK activation or c-Jun protein expression significantly reduced 1, 25(OH)(2)D(3)-induced alkaline phosphatase activity, a marker of CaCo-2 cell differentiation, in secosteroid-treated cells. Taken together, the present study demonstrated that 1,25(OH)(2)D(3) stimulated AP-1 activation in CaCo-2 cells by a PKC-alpha- and JNK-dependent mechanism leading to increases in cellular differentiation.
Caco-2 Cells↗
UV irradiation activates JNK and increases alphaI(I) collagen gene expression in rat hepatic stellate cells.
Hepatic stellate cells (HSCs) become activated into myofibroblast-like cells during the early stages of hepatic injury associated with fibrogenesis. The subsequent dysregulation of alphaI(I) collagen gene expression is a central pathogenetic step during the development of cirrhosis. Our recent study in rat HSCs (Davis, B. H., Chen, A., and Beno, D. (1996) J. Biol. Chem. 271, 11039-11042) found that ERK1,2 activation might be required for maximal alphaI(I) collagen gene expression. However, the role of the parallel JNK cascade in regulating alphaI(I) collagen gene expression was unknown. In this study, we initially found that UV irradiation of HSCs activated JNK but not ERK1,2. Furthermore, UV irradiation increased endogenous alpha I(I) collagen mRNA abundance and stimulated alpha I(I) collagen gene transcription in HSCs. The effect of the activation of JNK and Jun on alpha I(I) collagen gene expression was further evaluated via transfection of chloramphenicol acetyltransferase reporter plasmids with various sizes of truncated 5' upstream promoter sequence (UPS) of the alphaI(I) collagen gene. This revealed that dominant negative transcription factor JUN suppressed alpha I(I) collagen gene transcription in HSCs maintained in media with 20% serum and constitutively activated JUN increased alphaI(I) collagen gene transcription in HSCs cultured in media with 0.4% serum. UV activated JNK utilized a distal GC box in the 5'-UPS of the collagen gene to regulate gene transcription. This observation was confirmed by site-directed mutagenesis. In co-transfection experiments, the col-chloramphenicol acetyltransferase reporter with a mutagenized GC box was not suppressed by dn-JUN and was not stimulated by activated JUN or by UV irradiation. Southwestern blotting analyses and gel shift assays with basic transcription element-binding protein antiserum suggested that the GC box was bound by basic transcription element-binding protein, a recently described DNA-binding protein. In conclusion, the current study combined with our previous report suggests that ERK1,2 and JNK cascades regulate alphaI(I) collagen expression in HSCs through different regions of the 5'-UPS of the gene. The distal GC box in the 5'-UPS of the alphaI(I) collagen gene may play a central role in receiving extracellular signals through the JNK pathway.
Animals↗
Metabolic oxidative stress activates signal transduction and gene expression during glucose deprivation in human tumor cells.
The mechanism of glucose deprivation-induced activation of Lyn kinase (Lyn), c-Jun N-terminal kinase 1 (JNK1) and increased expression of basic fibroblast growth factor (bFGF) and c-Myc was investigated in MCF-7/ADR adriamycin-resistant human breast carcinoma cells. Glucose deprivation significantly increased steady state levels of oxidized glutathione content (GSSG) and intracellular prooxidants (presumably hydroperoxides) as well as caused the activation of Lyn, JNK1, and the accumulation of bFGF and c-Myc mRNA. The suppression of GSSG accumulation and prooxidant production by treatment with the thiol antioxidant, N-acetylcysteine, also suppressed all the increases in kinase activation and gene expression observed during glucose deprivation. In addition, glucose deprivation was shown to induce oxidative stress in IMR90 SV40 transformed human fibroblasts, indicating that this phenomena is not limited to the MCF-7/ADR cell line. These and previous observations from our laboratory show that glucose deprivation-induced oxidative stress in MCF-7/ADR cells activates signal transduction involving Lyn, JNK1, and mitogen activated protein kinases (ERK1/ERK2) which results in increased bFGF and c-Myc mRNA accumulation. These results provide support for the hypothesis that alterations in intracellular oxidation/reduction reactions link changes in glycolytic metabolism to signal transduction and gene expression in these human tumor cells.
Fibroblast Growth Factor 2↗
E-box-binding repressor is down-regulated in hepatic stellate cells during up-regulation of mannose 6-phosphate/insulin-like growth factor-II receptor expression in early hepatic fibrogenesis.
Hepatic stellate cells become activated during the early stages of hepatic injury associated with fibrogenesis. The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGFIIR) plays an important role in early fibrogenesis by participating in the activation of latent transforming growth factor-beta, a potent inducer of the matrix proteins in activated stellate cells that define the fibrotic phenotype. In this study we examined hepatic stellate cell regulation of M6P/IGFIIR expression and found that M6P/IGFIIR mRNA transcript levels increased in stellate cells from rats exposed to carbon tetrachloride (CCl4), a potent fibrogenic stimulant. Two E-boxes residing in the proximal promoter of M6P/IGFIIR were found to each bind a novel 75-kDa transcription factor (P75) in quiescent stellate cells of normal livers. This E-box binding was down-regulated as an early response in stellate cells exposed to CCl4, coinciding with increased M6P/IGFIIR transcript levels. Mutagenized E-boxes in M6P/IGFIIR promoter-chloramphenicol acetyltransferase (CAT) reporter constructs produced a substantial increase in reporter expression when compared with the corresponding native promoter-CAT construct when transfected in culture-activated stellate cells, suggesting P75's role as a repressor. The results indicate P75's participation in the regulation of M6P/IGFIIR transcription in hepatic stellate cells during fibrogenesis.
Animals↗
Decreased PKC-alpha expression increases cellular proliferation, decreases differentiation, and enhances the transformed phenotype of CaCo-2 cells.
Previous studies have shown that PKC-alpha protein expression is decreased in sporadic human colon cancers, as well as in colonic tumors of rats induced by chemical carcinogens. To elucidate the potential role of PKC-alpha on several phenotypic characteristics of colon cancer cells, we have transfected cDNAs for PKC-alpha in sense or antisense orientations into CaCo-2 cells, a human colonic adenocarcinoma cell line. Transfected clones were isolated that demonstrated approximately 3-fold increases (sense transfectants) and approximately 95% decreases (antisense transfectants) in PKC-alpha expression with no significant alterations in other PKC isoforms. Transfection of CaCo-2 cells with PKC-alpha in the antisense orientation resulted in enhanced proliferation and decreased differentiation, as well as in a more aggressive transformed phenotype compared with empty vector-transfected control cells. In contrast, cells transfected with PKC-alpha cDNA in the sense orientation demonstrated decreased proliferation, enhanced differentiation, and an attenuated tumor phenotype compared with these control cells. These data show that alterations in the expression of PKC-alpha induce changes in the proliferation, differentiation, and tumorigenicity of CaCo-2 cells. Furthermore, these findings indicate that loss of PKC-alpha expression in sporadic human and chemically induced colonic cancers may confer a relative growth advantage during colonic malignant transformation.
Animals↗