BackgroundPoly-ADP-ribosylation, a reversible post-translational modification of primarily chromosomal proteins, is involved in various cellular and molecular processes including carcinogenesis. ADP-ribose polymer or poly-ADP-ribose adducts are enzymatically added onto or stripped off the target chromosomal proteins during this metabolic process. Due to this, the chromatin superstructure is reversibly altered, which significantly influences the pattern of gene expression. We hypothesize that a decrease in the concentration of total poly-ADP-ribose adducts of peripheral blood lymphocyte (PBL) proteins strongly correlates with the incidence of human cancer.ResultsUsing a novel immunoprobe assay, we show a statistically significant (P ≤ 0.001) reduction (~ 42 to 49%) in the level of poly-ADP-ribose adducts of PBL proteins of patients with advanced cancers of head & neck (H & N) region (comprising fourteen distinct cancers at different sites), breast and cervix in comparison to healthy controls.ConclusionsThese findings imply potential utility of the poly-ADP-ribose adducts of PBL proteins as a novel and general biomarker of human cancers with potentials of significant clinical and epidemiological applications.
Background Immunofluorescence techniques done on formalin-fixed, paraffin-embedded tissue can serve as salvage techniques in cases where immunofluorescence on the frozen section may not be adequate or available. The present study was undertaken to assess the diagnostic utility of paraffin immunofluorescence by proteinase K digestion on renal biopsy compared to fresh frozen immunofluorescence. Methodology The paraffin immunofluorescence by proteinase K digestion of paraffin-embedded renal biopsy (IF-FFPE) was standardized and compared with the immunofluorescence on fresh frozen tissue (IF-Frozen). A total of 50 cases of the native renal biopsy were included in the study, and their intensity for fluorescein isothiocyanate-labeled IgA, IgG, IgM, C3, kappa, and lambda was compared. Results A total of 50 cases of the native renal biopsy were included in the study, and their intensity for fluorescein isothiocyanate-labeled antibodies of IgA, IgG, IgM, C3, kappa, and lambda was compared. The difference of 2+ intensity of antibodies between IF-FFPE and IF-Frozen was noted mainly in lupus nephritis (15%), followed by IgA nephropathy (10%) and membranoproliferative glomerulonephritis (7%). IF-FFPE showed a sensitivity of 90.3%, 91.8%, 82.7%, 81.1%, 92.1%, and 94.6% for IgA, IgG, IgM, C3, kappa, and lambda, respectively, whereas specificity was 100% for IgA, IgG, C3, kappa, and lambda and 95.2% for IgM. Conclusions Immunofluorescence techniques done on formalin-fixed, paraffin-embedded tissue can serve as salvage techniques in kidney biopsies.
Background: Poly-ADP-ribosylation (PAR), a reversible post-translational modification of cellular proteins, is involved in various cellular and molecular processes including carcinogenesis (Althaus & Richter, 1987; Jacobson & Jacobson, 1989; Poirier & Moreau, 1992; Boulikas, 1993; Sharan, 2009). ADP-ribose polymer adducts are added onto target cellular proteins by this metabolic process. We hypothesize that a decrease in the concentration of total poly-ADP-ribose adducts of peripheral blood lymphocyte (PBL) proteins strongly correlates with incidence of human cancer. Methods: A novel, sensitive and convenient immunoprobe assay of poly-ADP-ribose adducts of cellular proteins has been employed using slot blots of total PBL lysate (Sharan et al, 1998; 2005; Sharan, 2009). This assay quantifies the metabolic concentration of poly-ADP-ribose adducts of cellular proteins. Results: Using the immunoprobe assay, we show a statistically significant (P ≤ 0.001) reduction (~ 42 to 49%) in the level of poly-ADP-ribose adducts of PBL proteins of patients with advanced cancers of head & neck (H & N) region (comprising fourteen distinct types of cancers at different sites), breast and cervix in comparison to healthy controls. Conclusion: These preliminary findings may imply potential utility of the poly-ADP-ribose adducts of PBL proteins as a novel and general biomarker of human cancers with potentials of clinical and epidemiological applications.
Poly-ADP-ribosylation (PAR),Previous detailed investigations done in murine model using the novel immunoprobe assay have shown that the PAR of cellular proteins is significantly reduced during carcinogenesis 7, 8, 9, 10,11,12,13 . We hypothesize that these results could be applied to human cancer scenario. In order to test the hypothesis we have detected and quantified the total PAR of PBL proteins in cancer patients and healthy individuals.PBL was chosen since (a) PBL proteins have been shown to mirror status of PAR of cellular proteins of liver, spleen, ascites cells and other tissues 9 and (b) a blood based assay of PAR would be the most non-invasive and easy one to perform on humans.An assay using other tissues or biopsies pose practical problems as obtaining the tissue requires surgical intervention. Blood, being a circulating tissue, would be sentinel of the whole body and is, therefore, likely to be an ideal target tissue for cancer detection program 14 .Blood samples were collected from new cancer patients (cases), who had not volunteers with no known history of cancer (age range 25-80 years). Analysis of age distribution of controls and cases was done using unpaired t-test with Welch correction and χ 2 test, which showed that statistically older males (P ≤ 0.0001), but not females (P = 0.4250), made the case group ( Fig. 1 -Table). Overall, the cancer patients (cases) were statistically older (47.70 years) than the control counterparts (39.90 years) ( Fig. 1 -Table). The PBL protein samples (500 ng per slot) were prepared from blood samples of controls (n = 68) and cases (total n = 112). The cases comprised patients with cancers of 14 different sites grouped as cancer of H & N (total n = 66), breast (n = 22) or cervix (n = 24). Samples were subjected to quantitative detection of PAR using the novel slot blot immunoprobe assay followed by densitometric analysis as described 7 . Figure 1 -a shows a typical slot blot stained P a g e | 3for total proteins using India ink (left panel) and its immunoprobed replicum (right panel) for five replicate samples from a control subject (A) and a cancer patient (B).The concentration corrected mean net intensity of each control sample has been plotted as a dot to show the spread of measured PAR levels in relation to sample number ( Fig. 1 -b), age of patient ( Fig. 1 -c) and gender of patient ( Fig. 1 -d) highlight the range of variation in measured control values in arbitrary units (AU) (mean: 14573±1452 AU; range: 11950 -17540 AU). Compared to the control group, the total PAR of PBL proteins of H & N cancer patients showed a statistically significant (P <0.0001) reduction in PAR for cancers of all 14 sites (Fig. 2 -a , Table). The overall reduction was approximately 45% with a mean of net intensity of 8060±707.10 AU (range of 6324 -10318 AU). Cancers of all sites in this groupindividually also showed statistically significant reductions (see supplementary materials, Table I). Similarly, there were significant reductions in the total PAR of PBL proteins in cancers o...
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