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ABSTRACT: The purpose of this study was to determine if increased NF-κB activity of highly invasive PC-3 cells contributed to their
invasive behavior. Increased NF-κB activity has been observed in several malignant tumors and it may have an important role
in tumorigenesis, progression and chemotherapy resistance. By serial selection, we obtained invasion variant PC-3 cell sublines.
The PC-3 High Invasive cells invade readily through a Matrigel® reconstituted basement membrane while PC-3 Low Invasive cells have low baseline invasion activity. In these studies, we discovered
that NF-κB DNA binding activity was increased in PC-3 High Invasive cells when compared to PC-3 Low Invasive cells by electrophoretic
mobility shift assay (EMSA). Gel supershift assays showed a 4-fold increase in p65 containing complexes and a 2.2-fold increase
in the p50 containing complexes in the PC-3 High Invasive cells. Luciferase reporter assays showed that NF-κB dependent transcription
activity was increased 10.2±2.5-fold in the highly invasive cells (P<0.002). The PC-3 High Invasive cells showed a constitutive increase in phospho-IκBα and introduction of the super-repressor
IκBα S32/36A inhibited NF-κB activity to 19.2±2.5 percent of control transfected cells (P≤0.001). The IκBα super-repressor reduced the basement membrane invasion of PC-3 High Invasive cells from 6.2±1.1 to 3.8±0.4
percent (P<0.002) with no decrease in cell viability or proliferation. These results demonstrate that increased NF-κB activity contributed
directly to the invasive behavior of PC-3 High Invasive prostate cancer cells.
Clinical and Experimental Metastasis 04/2012; 18(6):471-479. · 3.52 Impact Factor
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ABSTRACT: Conventional Fourier-transform infrared (FTIR) microspectroscopic systems are limited by an inevitable trade-off between spatial resolution, acquisition time, signal-to-noise ratio (SNR) and sample coverage. We present an FTIR imaging approach that substantially extends current capabilities by combining multiple synchrotron beams with wide-field detection. This advance allows truly diffraction-limited high-resolution imaging over the entire mid-infrared spectrum with high chemical sensitivity and fast acquisition speed while maintaining high-quality SNR.
Nature Methods 03/2011; 8(5):413-6. · 19.28 Impact Factor
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ABSTRACT: The enzyme arylsulfatase B (N-acetylgalactosamine-4-sulfatase; ARSB; ASB) removes 4-sulfate groups from the sulfated glycosaminoglycans (sGAG) chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Inborn deficiency of ARSB leads to the lysosomal storage disease mucopolysaccharidosis VI, characterized by accumulation of sGAG in vital organs, disruption of normal physiological processes, severe morbidity, and premature death. Recent published work demonstrated extra-lysosomal localization with nuclear and cell membrane ARSB observed in bronchial and colonic epithelial cells, cerebrovascular cells, and hepatic cells. In this report, the authors present ARSB immunostaining in a colonic microarray and show differences in distribution, intensity, and pattern of ARSB staining among normal colon, adenomas, and adenocarcinomas. Distinctive, intense luminal membrane staining was present in the normal epithelial cells but reduced in the malignancies and less in the grade 3 than in the grade 1 adenocarcinomas. In the normal cores, a distinctive pattern of intense cytoplasmic positivity at the luminal surface was followed by reduced staining deeper in the crypts. ARSB enzymatic activity was significantly greater in normal than in malignant tissue. These study findings affirm extra-lysosomal localization of ARSB and suggest that altered ARSB immunostaining and reduced activity may be useful indicators of malignant transformation in human colonic tissue.
Journal of Histochemistry and Cytochemistry 03/2011; 59(3):328-35. · 2.72 Impact Factor
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ABSTRACT: Tissue factor (TF) is a cell surface glycoprotein intricately related to blood coagulation and inflammation. This study was performed to investigate the role of monocyte-lineage cells in prostate cancer cell TF expression and cell invasion.
Prostate cancer cell invasion was tested with and without added peripheral blood monocytes or human monocyte-lineage cell lines. TF neutralizing antibodies were used to determine the TF requirement for prostate cancer cell invasion activity. Immunohistochemistry was performed to identify prostate tissue CD68 positive monocyte-derived cells and prostate epithelial TF expression.
Co-culture of PC-3, DU145, and LNCaP cells with isolated human monocytes significantly stimulated prostate cancer cell invasion activity. TF expression was greater in highly invasive prostate cancer cells and was induced in PC-3, DU145, and LNCaP cells by co-culture with U-937 cells, but not with THP-1 cells. TF neutralizing antibodies inhibited PC-3 cell invasion in co-cultures with monocyte-lineage U-937 or THP-1 cells. Prostate cancer tissues contained more CD68 positive cells in the stroma and epithelium (145 ± 53/mm(2)) than benign prostate (108 ± 31/mm(2)). Samples from advanced stage prostate cancer tended to contain more CD68 positive cells when compared with lower stage lesions. Prostatic adenocarcinoma demonstrated significantly increased TF expression compared with benign prostatic epithelium.
This study shows that co-culture with monocyte-lineage cells induced prostate cancer cell invasion activity. PC-3 invasion and TF expression was induced in co-culture with U-937 cells and partially inhibited with TF neutralizing antibodies.
The Prostate 11/2010; 70(15):1672-82. · 3.48 Impact Factor
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ABSTRACT: There are very limited data concerning the association of lymphocytic thyroiditis with cardiomyopathy, and there is only one published report of fatal cardiomyopathy associated with autoimmune thyroiditis. An association of Hashimoto encephalopathy (HE) with autoimmune myocarditis has not yet been reported. Here we describe a case of a 31-year-old man with autoimmune thyroiditis complicated by HE, who succumbed to autoimmune myocarditis. This association raises the possibility that HE and myocarditis may share a common pathogenetic mechanism in some cases.
Pathology - Research and Practice 10/2010; 206(10):720-2. · 1.21 Impact Factor
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ABSTRACT: When clinicians contemplate the use of a new predictive technology in their practice, such as a nomogram, there is always a question of whether the new test is beneficial to their own clinical population. Unfortunately, traditional validation methods require a large number of subjects for validation testing and delay the decision-making process. We present an efficient and easy-to-use method based on the concept of sequential data analysis.
We illustrate with an example determining the validity of a technology for predicting Gleason score upgrading from biopsy to postprostatectomy (the Chun nomogram) in a clinical population different from the one used to initially validate the technology. Clinical data required by the Chun nomogram were available from 201 patients from the Cooperative Prostate Cancer Tissue Resource.
Of 124 patients predicted by the Chun nomogram to have an upgrading event, 47 actually did. The positive predictive value (PPV) of the model was therefore 38% and significantly (P < .05) less than the value of 80% which we considered to be the smallest clinically useful PPV in this situation. Had the sequential methods introduced in this article been employed prospectively in this cohort, the same conclusion would have been reached using data from only the first 15 patients.
In-clinic validation of predictive technologies will help the clinician adopt truly beneficial technologies and avoid the adoption of technologies which provide no significant benefit to their local patient population. For this task, sequential methods offer clear advantages.
Journal of Clinical Oncology 01/2009; 27(7):1087-90. · 18.37 Impact Factor
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ABSTRACT: Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a valuable and abundant resource of pathologic material for various biomedical studies. In the present study, we report the application of high-resolution inductively coupled mass-spectrometry (ICP-MS) for quantification of Fe, Zn, Se and Cd in FFPE prostate tissue. These elements have a possible role in the development of prostate diseases: while Zn and Se are needed for a healthy prostate, Cd shows multiple toxic and carcinogenic effects. Excessive accumulation of Fe induces the production of highly reactive hydroxyl radical species, which may play a role in cancer etiopathogenesis. To assess whether the levels of these metals in the FFPE prostate tissue represent their original content, we compared their levels with those in the fresh tissue (on dry weight basis) in samples obtained from 15 patients. We found that in FFPE tissue, the recoveries of Se, Fe, Cd and Zn were progressively decreased, 97+/-11% (r=0.88), 82+/-22% (r=0.86), 59+/-23% (r=0.69) and 24+/-11% (r=0.38), respectively. Thus, the use of correction factors, determined as k=0.16 for Se, k=0.20 for Fe, k=0.27 for Cd and k=0.67 for Zn, is required to estimate the retrospective levels of these elements in the parental non-processed fresh (wet) prostate tissue. The technique used in this study enables the analysis of archival FFPE prostate tissue for the concentrations of Fe, Zn, Se and Cd to study association between the levels of these metals and prostate disease.
Journal of Trace Elements in Medicine and Biology 02/2008; 22(4):305-14. · 1.68 Impact Factor
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ABSTRACT: Immunohistochemical staining with alpha-methylacyl-CoA racemase AMACR (P504S) has been described in a number of normal tissues and was found to be useful for detecting malignancies including hepatocellular carcinoma (HCC). Our aim was to determine whether AMACR is differentially expressed in benign nondysplastic liver tissue, hepatocellular dysplasia, and HCC. The study material consisted of paraffin blocks containing primary HCC and surrounding liver tissue from 20 patients who underwent hepatectomy at the time of liver transplantation. Immunohistochemical stains were performed with anti-AMACR by standard methods. Staining features were characterized on the basis of the pattern and distribution of reactivity. A positive AMACR immunostain was defined as either finely stippled or coarsely granular in pattern, in a diffuse or parabasal cytoplasmic distribution. A negative AMACR immunostain was defined as absence of reactivity. Anti-AMACR immunostains were positive in malignant, dysplastic, and benign nondyplastic hepatocytes in all cases. The staining pattern was the same in malignant and dysplastic hepatocytes. It consisted of coarsely granular reactivity in a parabasal or diffuse cytoplasmic distribution. In contrast, benign nondysplastic hepatocytes were distinguished by weak, finely stippled diffuse cytoplasmic staining. Malignant and dysplastic hepatocytes showed an identical pattern of immunostaining for AMACR that was distinct from benign hepatocytes. Prospective studies are needed to determine whether staining for AMACR can distinguish HCC or dysplasia in cytologic and small histologic specimens.
Applied immunohistochemistry & molecular morphology: AIMM / official publication of the Society for Applied Immunohistochemistry 01/2007; 14(4):411-6. · 1.63 Impact Factor
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ABSTRACT: Endometriosis, defined as the presence of endometrial glandular and stromal cells outside the uterine cavity, is a common gynecological disease with poorly understood pathogenesis. Using laser capture microdissection and a cDNA microarray with 9600 genes/expressed sequence tags (ESTs), we have conducted a comprehensive profiling of gene expression differences between the ectopic and eutopic endometrium taken from 12 women with endometriosis adjusted for menstrual phase and the location of the lesions. With dye-swapping and replicated arrays, we found 904 genes/ESTs that are differentially expressed. We validated the gene expression using real-time RT-PCR. We found that the expression patterns of these genes/ESTs correctly classified the 12 patients into ovarian and nonovarian endometriosis. We identified gene clusters that are location-specific. In addition, we identified several biological themes using Expression Analysis Systematic Explorer. Finally, we identified 79 pathways with over 100 genes with known functions, which include oxidative stress, focal adhesion, Wnt signaling, and MAPK signaling. The identification of these genes and their associated pathways provides new insight. Our findings will stimulate future investigations on molecular genetic mechanisms underlying the pathogenesis of endometriosis.
Endocrinology 02/2006; 147(1):232-46. · 4.46 Impact Factor
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ABSTRACT: To test the hypothesis that endometriosis may originate from genomic alterations in the endometrium by genomic analysis of endometrial tissues in patients with endometriosis and compare them with those from normal controls.
Endometrial tissue samples were taken from five women with endometriosis. For controls, we used endometrial tissue samples from four women who underwent elective abortions and one sample from placenta. Using array-based comparative genomic hybridization (CGH), we determined the normal range of variation in CGH signals using normal controls. CGH results were further confirmed by real-time quantitative PCR and loss of heterozygosity analysis.
We identified several regions of genomic alterations in all five patients. Some of these regions were the same regions identified previously in endometriotic lesions. For select markers, the genomic alterations were confirmed by real-time PCR and LOH analyses.
There is evidence that the endometrium in women with endometriosis has genomic alterations. This is consistent with numerous reports that the endometrium of women with endometriosis differ from those of women without. Our finding suggests that genomic alterations in the endometrium may be a proximate cause for endometriosis.
European Journal of Obstetrics & Gynecology and Reproductive Biology 10/2004; 116(1):89-99. · 1.97 Impact Factor
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ABSTRACT: To determine the clonal origins of endometriotic lesions using laser capture microdissection and PCR-based HUMARA assay.
Molecular genetic study of human tissue.
Molecular genetics laboratory in an academic setting.
Twenty patients with endometriosis. Forty specimens of endometriotic lesions from these patients and one specimen of normal endometrium were analyzed.
Laser capture microdissection was used to harvest epithelial cells from single and multifocal endometrial lesions from paraffin-embedded and frozen tissues, and their clonality was determined with the HUMARA assay.
Polymerase chain reaction-based HUMARA assay of clonality.
Thirty-eight specimens were polymorphic and thus informative. Most specimens were monoclonal, as determined by the HUMARA assay. In four specimens of multifocal lesions, polyclonality was detected, but upon more refined microdissections and further analyses, we found that each focus was monoclonal individually.
Previously reported polyclonality is very likely to be attributed to the pooling of multifocal lesions or contamination of normal tissues. These results suggest that endometriotic lesions were monoclonal in origin, and in the case of multifocal lesions, each focus originates monoclonally; hence, different foci have independent origins. The monoclonality of endometriotic lesions suggests that they may carry neoplastic potentials, and the apparent independent origins of multifocal lesions suggest that reconstruction of individual lesion histories may help us to understand the initiation and progression of endometriosis.
Fertility and Sterility 04/2003; 79 Suppl 1:710-7. · 3.56 Impact Factor
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ABSTRACT: To determine the molecular mechanisms of aggressive prostate cancer behavior, we studied RhoGTPases in high and low invasive variants of PC-3 prostate cancer cells. Prior studies with these cells revealed that elevated nuclear factor kappaB (NF-kappaB) expression and activity were necessary for the highly invasive phenotype. In the current study, increased RhoA expression was found in the PC-3 highly invasive cells as compared with the PC-3 low invasive cells through cDNA array and Western blot analyses. Similarly, RhoA activity, as measured by the Rhotekin binding assay, was elevated in the PC-3 highly invasive cells. Transfection of these highly invasive cells with dominant negative RhoA N19 or treatment with 1.0 micro g/ml RhoA inhibitor C3 exoenzyme demonstrated that RhoA activity was necessary for both NF-kappaB activity and cellular invasion of a Matrigel reconstituted basement membrane. Furthermore, stable transfection of the PC-3 highly invasive cells with constitutively active RhoA Q63L resulted in activation of NF-kappaB activity and Matrigel invasion, effects reversed by treatment of the cells with C3 exoenzyme. RhoA was also shown to act through the motility component of the invasion process. RhoA activity was therefore both necessary and sufficient for the elevated NF-kappaB, invasion, and motility activities of the PC-3 highly invasive cells. These findings suggest molecular targets to control cancer cell invasion and aid in the development of definitive tools for predicting the invasive and metastatic potential of cancer cells.
Cancer Research 04/2003; 63(6):1359-64. · 7.86 Impact Factor
