Hyperproliferative Activity Research Articles

Abstract 10: Development of single cell spheroid as a tool to capture functional heterogeneity in head and neck cancer

Abstract Introduction: Head and neck cancer squamous cell carcinomas (HNSCC) are diverse and complex with inter-tumor and intra-tumor heterogeneity and discrepancies in therapy response regardless of the clinical stage. Tumors are thought to be heterogeneous at the single-cell level involving cancer stem cells. Therefore, cancer diagnosis and treatment are difficult because single cells might have intrinsic functional and genetic heterogeneity. Single cell transcriptome is used for mapping the genetic heterogeneity of a tumor. Currently, no model is available to provide a genotype to phenotype correlations for the heterogeneity, a crucial step for successful treat of a cancer. Here, our focus is to develop a single-cell clonal spheroid model system as a valuable tool for studying functional heterogeneity, including growth, proliferation, angiogenesis, metastasis, and genetic heterogeneity. Material and methods: HNSCC cell lines namely, UMSCC-22B, FaDu and A-253, and patients derived primary cells were cultured in 96-well ultra-low attachment to develop spheroids. Spheroids were cultured from 2, 4 and 128 cells per well with media, growth factors and supplements. Confocal microscopy was done using cocktail dyes including Hoechst, Calcein-AM and Ethidium bromide to check the functionality of spheroids. Results: We have established a method for the development of single-cell derived clonal spheroids. The frequency of spheroids formation from 2, 4 and 128 UMSCC-22B cells per well were higher than FaDu and A-253 cell lines. The growth measurement showed clones of single cell derived spheroids with hyperproliferation as well as with low proliferative activity. UMSCC-22B and A-253 hypoproliferative spheroids had significant higher migration capabilities compared to spheroids with hyperproliferative activity. Confocal microscopy of single-cell derived clonally propagated spheroids revealed a different zone viz proliferating, quiescent and necrotic zone in intact spheroids. This condition mimics in vivo conditions and suitable tool for drug penetration studies. Conclusion: Together, our results suggest that single cell-derived clonal spheroid from different wells would represent the genetic and functional heterogeneity of the cell population. Such a spheroid model can reveal heterogeneity inside a tumor formed by the proliferation of a single-cell and heterogeneity between tumors resulting from a different single cell and thus has great potential for drug discovery and tumor biology research. Citation Format: Jyoti Pandey, Ritis Shyanti, Sanjay V. Malhotra, Rupesh Chaturvedi, Rana P. Singh. Development of single cell spheroid as a tool to capture functional heterogeneity in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 10.

Nintedanib inhibits keloid fibroblast functions by blocking the phosphorylation of multiple kinases and enhancing receptor internalization.

Keloid is a benign skin tumor characterized by its cell hyperproliferative activity, invasion into normal skin, uncontrolled growth, overproduction and deposition of extracellular matrices and high recurrence rate after various therapies. Nintedanib is a receptor tyrosine kinase inhibitor targeting VEGF, PDGF, FGF, and TGF-β receptors with proved efficacy in anti-angiogenesis and in treating various types of cancers. In this study, we investigated the effects of nintedanib on keloid fibroblasts in both in vitro and ex vivo models. Keloid fibroblasts were prepared from 54 keloid scar samples in active stages collected from 49 patients. We found that nintedanib (1−4 μM) dose-dependently suppressed cell proliferation, induced G0/G1 cell cycle arrest, and inhibited migration and invasion of keloid fibroblasts. The drug also significantly inhibited the gene and protein expression of collagen I (COL-1) and III (COL-3), fibronectin (FN), and connective growth factor (CTGF), as well as the gene expression of other pathological factors, such as alpha smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), FK506-binding protein 10 (FKBP10), and heat shock protein 47 (HSP47) in keloid fibroblasts. Furthermore, nintedanib treatment significantly suppressed the phosphorylation of p38, JNK, ERK, STAT3, and Smad, enhanced endocytosis of various growth factor receptors. Using an ex vivo tissue explant model, we showed that nintedanib significantly suppressed cell proliferation, migration, and collagen production. The drug also significantly disrupted microvessel structure ex vivo. In summary, our results demonstrate that nintedanib is likely to become a potential targeted drug for keloid systemic therapy.

Clinical Heterogeneity of AML Is Associated with Mutational Heterogeneity

BACKGROUND: AML is a group of clinically heterogeneous diseases. We hypothesized that heterogeneous presentation of AML is a reflection of equally heterogeneous genetic process during the leukemogenesis.METHODS: 536 AML patients (pts) bone marrow samples were analyzed by targeted capture exome sequencing of 295 genes. Extensive clinical-genotype correlation was performed using well annotated clinical data.RESULTS: The median age of the cohort was 62 years (IQR: 51-72) including 297 (55%) elderly (age ≥60), and 239 (45%) young (age <60) pts. Of the 536 pts, 308 (57%) pts had de novo AML (dnAML), and 103 (19%) had secondary or therapy-related AML (stAML). DNA sequencing revealed 1,586 high-confidence somatic mutations (922 SNVs and 664 indels) in 79 genes in 493 (92%) pts [median 3 (IQR 2-4) mutations/patient]. Cytogenetics were favorable in 10 (2%), intermediate in 326 (61%), and adverse in 177 (33%) (All defined by ELN 2017criteria); 23 (4%) pts had no cytogenetics data.Elderly pts and young pts had distinct mutational landscape. (1.3-9.6), p = 0.0079] were significantly more enriched in elderly AML, whereas young AML pts were enriched with mutations in FLT3 [OR 0.6 (0.4-0.9), p = 0.0089], NPM1 [OR 0.5 (0.3-0.9), p = 0.0113], PTPN11 [OR 0.2 (0.2-0.7), p = 0.0033], and WT1 [OR 0.4 (0.2-0.7), p = 0.0033]. Some of the mutations enriched in elderly pts are frequently observed in pts with clonal hematopoiesis with indeterminate potential. Based on the ontogeny of AML, PTPN11 [OR 7.6 (1-57.2), p=0.0210], NPM1 [OR 3.0 (1.5-6.1), p = 0.0007], WT1 [OR 2.9 (1.1-7.4), p=0.0279] mutations were significantly enriched in dnAML, while SF3B1 [OR 0.4 (0.18-0.89), p=0.0376], SRSF2 [OR 0.5 (0.3-0.85), p = 0.0109], TP53 [OR 0.5 (0.3-0.8), p = 0.0131], ASXL1 [OR 0.6 (0.36-0.95), p=0.0451] mutations were more enriched in stAML (Figure A).We then correlated mutation data with clinical and immunological parameters that are routinely tested in AML. Mutations in NPM1, FLT3, PTPN11 and NRAS were associated with significantly higher white blood cell (WBC) counts, bone marrow blast and LDH, which is consistent with their hyperproliferative activity as class 1 genes. In contrast, pts with mutations in TP53, STAG2 and ASXL1 presented with significantly low bone marrow blast, circulating blast, and WBC. Mutations in BCOR and ASXL1 was associated with significantly low LDH. Interestingly, pts with IDH2 mutations presented with significantly higher platelet, which is consistent with anecdotal report (DiNardo et al. Am J Hematology). Not surprisingly, TP53 mutations were associated with complex cytogenetics, whereas SRSF2, NPM1, IDH2, FLT3, and CEBPA mutations were associated with good and intermediate cytogenetics by ELN classification (Figure B). Pts with NPM1, IDH2, and IDH1 mutations were associated with less HLA-DR and CD34 expression in blast by flow cytometry. This is consistent with the frequent presentation of these AML sub-types with cuplike nuclei (Rakheja et al. BJH).DNA sequencing of a large cohort also allowed us to detect mutations that have not been as commonly reported in AML. We detected hot-spot mutations in exon 2 of MYC and MYCN genes in 9 (2%) AML pts. Additionally, internal tandem duplication (ITD) in MYC was also detected in one patient. Immunohistochemical staining showed that MYC expression was significantly elevated in patients with MYC mutations than in patients without the mutations (median H score 22 vs. 15 in MYC mutated vs. normal karyotype control, p < 0.001, 22 vs. 13.5 in MYC mutated vs. trisomy 8 control). These data suggest that a subset of AML is driven by the strong MYC signaling, consistent with a prior study (Ohanian et al. Leuk Lymphoma).CONCLUSION: Heterogeneous clinical presentation of AML has significant association with genetic heterogeneity, which suggest that distinct genetic basis of leukemogenic process has strong role in defining clinical presentation of AML. These data also help stratifying the patients for the likely target of precision medicine. DisclosuresDiNardo:Medimmune: Honoraria; Celgene: Honoraria; Agios: Consultancy; Abbvie: Honoraria; Karyopharm: Honoraria; Bayer: Honoraria. Kadia:Celgene: Research Funding; Pfizer: Consultancy, Research Funding; BMS: Research Funding; Jazz: Consultancy, Research Funding; Abbvie: Consultancy; Abbvie: Consultancy; Amgen: Consultancy, Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Takeda: Consultancy; Novartis: Consultancy; Celgene: Research Funding; Novartis: Consultancy. Cortes:novartis: Research Funding. Daver:Daiichi-Sankyo: Research Funding; Pfizer: Consultancy; Alexion: Consultancy; ARIAD: Research Funding; Karyopharm: Consultancy; ImmunoGen: Consultancy; Kiromic: Research Funding; Otsuka: Consultancy; Sunesis: Consultancy; Novartis: Research Funding; BMS: Research Funding; Incyte: Consultancy; Novartis: Consultancy; Sunesis: Research Funding; Karyopharm: Research Funding; Pfizer: Research Funding; Incyte: Research Funding. Pemmaraju:SagerStrong Foundation: Research Funding; celgene: Consultancy, Honoraria; cellectis: Research Funding; samus: Research Funding; daiichi sankyo: Research Funding; Affymetrix: Research Funding; stemline: Consultancy, Honoraria, Research Funding; plexxikon: Research Funding; novartis: Research Funding; abbvie: Research Funding.

Metabolism–epigenome crosstalk in physiology and diseases

The way in which energy is used in cells is determined under the influence of environmental factors such as nutritional availability. Metabolic adaptation is mainly achieved through the modulation of metabolic gene expression, and may also involve epigenetic mechanisms that enable long-term regulation. Recent studies have identified that nutrients and their metabolites exert an important influence on the epigenome, as they serve as substrates and/or coenzymes for epigenetic-modifying enzymes. Some epigenetic factors have been shown to regulate metabolic genes leading to a shift in energy flow. These findings suggest the concept of metabolism-epigenome crosstalk that may contribute to the formation of a long-term metabolic phenotype. This is particularly relevant to the pathogenesis of obesity and associated metabolic disorders, in which pre- and post-natal nutritional conditions affect disease risks in adulthood. Moreover, most cancer cells exploit metabolic pathways for their hyperproliferative activity, while metabolic misregulation leads to aberrant epigenetic regulation in some cancers. This review explores the possible mechanisms of metabolism-epigenome crosstalk that may facilitate our understanding of physiology and diseases.

CYTOKERATIN 16 EXPRESSION DOES NOT INDICATE CHOLESTEATOMA AGGRESSIVENESS

AimsCholesteatoma is characterised by a squamous epithelium in the middle ear and aggressive destructive behavior. A hyperproliferative epithelium also typically has an overexpression of cytokeratin 16 (CK16). The aim of this study was to compare CK16 expression in cholesteatoma epithelium and the epithelium of simple dry perforations.MethodsSamples were obtained from 60 patients: 30 with cholesteatoma and 30 with simple perforations. The epithelium was immunohistochemically stained for CK16. Correlations were made between the clinical picture and the absence or presence of complications in both groups.ResultsImmunohistochemical staining for CK16 was positive in 45% of specimens taken from the edge of simple perforations and in 51% of specimens taken from the cholesteatoma matrix. The difference was not statistically significant.ConclusionTwo implications of our findings may be suggested: in cases of cholesteatoma the change in CK expression from normal epithelium is not the only factor which makes cholesteatoma aggressive; alternatively, in normal tissue the edge of a simple perforation is not as stable as is thought so that at least 45% of perforations can show hyperproliferative activity. It is therefore unclear whether hyperproliferative activity in either cholesteatoma or a simple perforation is a marker of instability or of attempts at healing.

In vitro evaluation of novel chitosan derivatives sheet and paste cytocompatibility on human dermal fibroblasts

The cytocompatibility of novel chitosan derivative sheets and pastes were evaluated in vitro for possible utilization in wound dressing applications for wound healing. In this study, the cytotoxicity of oligo-chitosan (O-C), N, O-carboxymethyl-chitosan (NO-CMC) and N- carboxymethyl-chitosan (N-CMC) derivatives in sheet like and paste forms were evaluated using primary normal human dermal fibroblast cultures and hypertrophic scars; a fibrotic conditions representing a model of altered wound healing with overproduction of extracellular matrix and fibroblast hyperproliferative activity. Cytotoxicities of these chitosan derivatives were assessed using 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The results indicate that both chitosan derivative sheets and pastes have appropriate cytocompatibility and appear promising as safe biomaterials with potential wound healing applications. The SH120 (NO-CMC) derivatives sheet exhibited highest cytocompatibility property and may be regulated by MMP-13 in controlling the cell growth and its expression level.

Molecular pathways supporting the proliferation staging of malignant melanoma (Review)

The clinical diagnosis of cutaneous melanoma always calls for histological confirmation. In addition to the recognition of the classic aspects of the neoplasm, immunohistochemistry is determinant, in particular in the assessment of the size of the replicative compartment. Generally, the proliferation rate is indicative of the neoplastic progression and is related to the clinical growth rate of the neoplasm. It allows to distinguish high risk melanomas showing a high growth rate from those of lower malignancy associated with a restricted growth rate. In melanoma, the recruitment and progression of neoplastic cells in the cell cycle of proliferation have lost some of their controls that are normally processed by a series of key regulatory molecules. In addition, the apoptotic pathway counteracting any hyperproliferative activity is released of the dependency of specific regulated molecular mechanisms. This review summarizes the current knowledge on key molecular components involved in the deregulation of the growth fraction, cell proliferation and apoptosis in melanocytic neoplasms. The implication of cyclins and of the mitogen-activated protein kinase pathways are scrutinized. The involvement of neoplastic stem cells in the metastatic process is also discussed.

R445 – Expression of Proliferative Markers in Cholesteatoma

Problem Cholesteatoma is characterized by the presence of a squamous epithelium invading the middle ear altering its growth properties. This epithelium believed to have hyperproliferative properties. Inflammatory stimulation of the underlying connective tissue, as well as an autocrine mechanism, may be responsible for the dysregulation and abnormal proliferative feature of the keratinocytes in cholesteatoma. Methods Comparative investigations were performed to assess the epithelial cell kinetics of cholesteatoma and auditory meatal skin. Monoclonal antibody PCNA and Ki-67 immunohistochemical staining were applied. Results Specimens of cholesteatoma samples (n=30) showed an average PCNA score of 26.6% and an average Ki-67 score of 15.9%. Auditory meatal skin(n=8) revealed an average PCNA score of 8.2% and average Ki-67 score of 4.9%. The results of this study confirm a highly increase in the proliferation rate of cholesteatoma keratinocytes, which had an PCNA score that was 3.24 times higher than the score for keratinocyte of auditory meatal skin and Ki-67 score of cholesteatoma was 3.24 times higher than auditory meatal skin. Conclusion We conclude that cholesteatoma is a hyperproliferative activity and that PCNA and Ki-67 immunohistochemical staining are valuable tool for assessing cell kinetics in cholesteatoma. Significance The cholesteatoma possesses a variable degree of proliferative activities depending on its histologic composition. The formation and accumulation of the keratin due to the continuous epithelial growth probably are important elements in inducing the inflammation.

Cellular changes following direct vitamin D injection into the uraemia-induced hyperplastic parathyroid gland.

Background. Hyperplasia of the parathyroid gland (PTG) is associated not only with excessive secretion of parathyroid hormone (PTH) but also with changes in the parathyroid cell (PTC) characteristics (i.e. hyperproliferative activity and low contents of vitamin D and calcium-sensing receptors). The control of PTG hyperplasia is most important in the management of secondary hyperparathyroidism (SHPT), because the advanced stage of hyperplasia is considered irreversible. For the better control of the PTH level in dialysis patients with such advanced SHPT, percutaneous vitamin D injection therapy (PDIT) under ultrasonographic guidance was developed and various cellular changes caused by this treatment were also investigated using an animal model.Methods. The PTGs of Sprague–Dawley rats, which had been 5/6-nephrectomized and fed a high-phosphate diet, were treated with the direct injections of vitamin D agents, and cellular effects focusing the above-mentioned characters were investigated.Results. An adequacy of the direct injection technique into the rats’ PTGs and the successful effects of this treatment in various biochemical parameters were confirmed. Such characteristics of advanced SHPT were simultaneously improved; in particular, it was confirmed that this treatment may be effective in controlling PTG hyperplasia by, at least in part, apoptosis-induced cell death.Conclusions. A locally high level of vitamin D strongly may suppress PTH secretion and regress hyperplasia, which is involved in the induction of apoptosis in PTCs, based on the simultaneous improvements of cellular characters of advanced SHPT. The PTH control introduced by this treatment successfully ameliorated osteitis fibrosa (high bone turnover rate).

Inactivation of the Polycomb Group Protein Ring1B Unveils an Antiproliferative Role in Hematopoietic Cell Expansion and Cooperation with Tumorigenesis Associated with Ink4a Deletion

Polycomb group (PcG) proteins act as positive regulators of cell proliferation. Ring1B is a PcG gene essential for embryonic development, but its contribution to cell turnover in regenerating tissues in not known. Here, we have generated a conditional mouse mutant line to study the Ring1B role in adult hematopoiesis. Mutant mice developed a hypocellular bone marrow that paradoxically contained an enlarged, hyperproliferating compartment of immature cells, with an intact differentiation potential. These alterations were associated with differential upregulation of cyclin D2, which occurred in all mutant bone marrow cells, and of p16(Ink4a), observed only in the differentiated compartment. Concurrent inactivation of Ink4a rescued the defective proliferation of maturing cells but did not affect the hyperproliferative activity of progenitors and resulted in a shortening of the onset of lymphomas induced by Ink4a inactivation. These data show that Ring1B restricts the progenitors' proliferation and promotes the proliferation of their maturing progeny by selectively altering the expression pattern of cell cycle regulators along hematopoietic differentiation. The novel antiproliferative role of Ring1B's downregulation of a cell cycle activator may play an important role in the tight control of hematopoietic cell turnover.

Prostatic stromal cells derived from benign prostatic hyperplasia specimens possess stem cell like property

The hyper-proliferative activity of stromal smooth muscle (SM) cells is believed to be responsible for the pathogenesis of benign prostatic hyperplasia (BPH). We have observed that those stromal cells can differentiate into unrelated specialized cells. We thus hypothesize that stromal cells derived from adults prostate specimens may contain adult stem cells. To test this hypothesis, human prostate stromal primary cultures were established and used for characterization of their stem cell properties. Immunoblotting, immunohistochemistry, RT-PCR, and tissue culture techniques were used to characterize the primary cultured human prostate-derived stromal cells for their stem cell and differentiation properties. The plasticity of these stromal cells was analyzed using cell culture and histology techniques. Primary cultured prostate stromal cells from BPH patient possess polygonal and elongated fibroblast/myofibroblast cellular morphology. They are positive in CD30, CD34, CD44, NSE, CD133, Flt-1, stem cell factor (SCF), and neuron-specific enolase (NSE), but negative in C-Kit, stem cell antigen (SCA), SH2, CD11b. Expression of SM myogenic markers in these cells may be induced by sodium butyrate (NaBu) treatment. Induction to osteogenic and adipogenic differentiation in these cells is also evident. Our study on primary stromal cells from BPH patients have yielded many interesting findings that these prostate stroma cells possess: (1) mesenchymal stem cell (MSC) markers; (2) strong proliferative potential; and (3) ability to differentiate or transdifferentiate to myogenic, adipogenic, and osteogenic lineages. These cell preparations may serve as a potential tool for studies in prostate adult stem cell research and the regulation of benign prostatic hyperplasia.

Tumor Promotion and Oxidative Stress in Ferric Nitrilotriacetate–Mediated Renal Carcinogenesis: Protection by Adhatoda vasica

ABSTRACTIn the present study, we report the chemopreventive effects of Adhatoda vasica against ferric nitrilotriacetate (Fe-NTA)–induced renal oxidative stress, hyperproliferative response, and two-stage renal carcinogenesis. Fe-NTA (9 mg Fe/kg body weight, intraperitoneally) enhances renal lipid peroxidation, xanthine oxidase (XO), and hydrogen peroxide (H2O2) generation with concomitant reduction in renal glutathione content (GSH), antioxidant enzymes, and phase II metabolizing enzymes. It induces blood urea nitrogen, serum creatinine, ornithine decarboxylase (ODC) activity, and [3H] thymidine incorporation into renal DNA. It also enhances DEN (N-diethylnitrosamine)-initiated renal carcinogenesis by increasing the percentage incidences of kidney tumors. Pretreatment of rats orally with A. vasica (50 and 100 mg/kg body weight) resulted in a significant decrease in lipid peroxidation, H2O2 generation, xanthine oxidase (XO), blood urea nitrogen, serum creatinine, renal ODC activity, DNA synthesis (p < 0.001), and incidence of tumors. Renal GSH (p < 0.01), glutathione-metabolizing enzymes (p < 0.001), and antioxidant enzymes were also recovered significantly (p < 0.001). Thus, our results show that A. vasica may meet the criteria demanded from a chemopreventive agent and in a rodent system it can reduce hyperproliferative response toxicity and carcinogenic activity of Fe-NTA.

Expression of histo-blood group antigens in bronchial squamous metaplasia

The aim of the present study was to evaluate the expression of blood group antigens in squamous bronchial metaplasia in order to determine whether this factor could identify patients at risk of lung cancer. In total, 100 bronchial biopsies were included in the present study. The cases were classified according to the World Health Organization grading system. Immunohistochemical stains for histo-blood groups A and B, and reactivity tests to p53 and the cellular proliferation index were performed. A total of 56 (56%) patients belonged to blood group A. Among them, six (10.7%) patients who did not express antigen in squamous metaplasia, showed carcinoma at the moment of the biopsy (n = 3) or developed synchronous lung carcinoma (n = 3). A total of nine (9%) patients belonged to blood group B. Loss of antigenic expression was observed in five cases. All of them developed synchronous lung carcinoma. The patients with low- and high-grade dysplasia developed lung cancer in 71 and 100% of the cases, respectively. In conclusion, the findings of the present study suggest that the loss of histo-blood antigens expression is an event in the carcinogenesis of bronchial mucosa and it is usually associated with high-grade lesions and hyperproliferative activity.

Direct injection of calcitriol or its analog into hyperplastic parathyroid glands induces apoptosis of parathyroid cells

Hyperplasia of the parathyroid gland (PTG) is associated not only with excessive secretion of parathyroid hormone (PTH) but also with changes in the parathyroid cell (PTC) characteristics (i.e. hyperproliferative activity, and low contents of vitamin D and calcium-sensing receptors). Control of PTG hyperplasia is most important in the management of secondary hyperparathyroidism, but the advanced stage of hyperplasia is considered irreversible. In the present study, dialysis patients with PTG hyperplasia underwent direct injection of calcitriol or maxacalcitol (OCT) into the PTG. Ultrasonography showed that this treatment had significantly reduced PTG volume and tissue analysis using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and DNA electrophoresis indicated that cellular apoptosis had been induced. The mechanism of apoptosis was evaluated in detail in uremic rats fed a high-phosphate diet. OCT or its vehicle was directly injected into the rats' PTGs. In the PTGs treated by OCT, there was a significantly increased number of TUNEL-positive PTCs and DNA electrophoresis revealed the characteristic ladder pattern of DNA fragmentation, both findings indicative of apoptosis. There was also a significant upregulation of both vitamin D and Ca-sensing receptors in the PTCs and a clear shift of the Ca-PTH response curve to the left and downward. None of these findings was observed in the PTGs treated by vehicle. This novel treatment is successful in causing regression of PTG hyperplasia. Thus, it is expected to significantly reduce the PTH level and ameliorate the abnormal bone turnover and mineral metabolism.

Chemopreventive activity of glycyrrhizin on lead acetate mediated hepatic oxidative stress and its hyperproliferative activity in Wistar rats

Lead is a pervasive environmental pollutant with no beneficial biological role and its toxicity continues to be a major health problem due to its interference with natural environment. In the present study we have evaluated the chemopreventive effect of glycyrrhizin on lead acetate mediated hepatic oxidative stress, toxicity and tumor promotion related alterations in rats. Lead acetate (100 mg/kg bwt., i.p.) enhanced lipid peroxidation with concomitant reduction in glutathione, glutathione reductase, glutathione- S-transferase and glutathione peroxidase activities. There was an increase in the levels of transaminase enzymes and LDH. Lead acetate treatment also enhanced ornithine decarboxylase (ODC) activity and [ 3H] thymidine incorporation into hepatic DNA. Pretreatment of rats orally with glycyrrhizin (150 and 300 mg/kg bwt., orally) resulted in a significant decrease in hepatic microsomal lipid peroxidation ( P < 0.001) and increase in the level of GSH content ( P < 0.001) and its dependent enzyme. There was significant reduction in the levels of SGPT, SGOT and LDH ( P < 0.001). A significant inhibition in ODC activity and DNA synthesis ( P < 0.001) was also observed. On the basis of the above results it can be hypothesized that glycyrrhizin is a potent chemopreventive compound against lead acetate mediated hepatic oxidative stress, toxicity and tumor promotion related responses in rats.

NFκB-Inducing Kinase Deficiency Results in the Development of a Subset of Regulatory T Cells, which Shows a Hyperproliferative Activity upon Glucocorticoid-Induced TNF Receptor Family-Related Gene Stimulation

CD4+CD25+ regulatory T cells (T(reg)) play an important role in maintaining immunologic tolerance. Glucocorticoid-induced TNFR family-related gene (GITR) expressed preferentially at high levels on T(reg) has been shown to be a key player of regulating T(reg)-mediated suppression. A recent study reports that NF-kappaB-inducing kinase (NIK) expression in thymic stroma is important for the normal production of T(reg) but not for its suppression capacity. In this report, we have shown that T(reg) from NIK-deficient mice display hyperproliferative activities upon GITR stimulation through an IL-2-independent mechanism. Furthermore, high dose IL-2, anti-CD28 stimulation, or GITR ligand-transduced bone marrow-derived dendritic cells used as APC (culture conditions which drive T(reg) proliferation in vitro) could not ablate this difference in proliferative activity between NIK-deficient and wild-type T(reg). Additional experiments have shown NIK-deficient mice have a higher ratio of CD4+CD25+CD62L(low) T(reg) both in thymus and periphery than their wild-type littermates. This CD62(low) subset is responsible for the hyperproliferative activity upon GITR stimulation. These data suggest a novel role of NIK in controlling the development and expansion of CD4+CD25+ regulatory T cells.

The role of hyperplasia in multiple parathyroid adenomas.

Parathyroid adenoma is the most common cause of primary hyperparathyroidism (pHPT). Adenomas usually involve only a single gland, and the remaining glands are normal or suppressed. Multiple parathyroid adenomas have been reported to occur in as high as 11% of patients with pHPT. The significant incidence of multiple adenomas with histologic similarities to hyperplasia has raised the possibility that adenoma is a continuation of the hyperplasia state. To test this theory, we used molecular genetics to compare clonality and proliferative activity of parathyroid adenoma with its corresponding normal glandular tissue. Furthermore, we devised a scheme to definitively distinguish between the different parathyroid states on a molecular level, because histologic distinction is unreliable. The study included three patients with a diagnosis of singular parathyroid adenoma and three with double parathyroid adenomas. Paraffin-embedded surgical specimens of both adenomas and normal glands were retrieved from each patient. Clonal analysis of the phosphoglycerolkinase (PGK) gene has suggested that parathyroid adenomas are monoclonal. Clonality of parathyroid adenomas and normal parathyroid glands was studied by polymerase chain reaction-based restriction fragment length polymorphic analysis for the PGK gene. Proliferative activity of the specimens was also analyzed using the immunohistochemical markers PCNA and Ki-67. All adenomas were monoclonal and all normal parathyroid glands were polyclonal for the PGK gene in both the single and double adenoma specimens. All adenomas stained positive for proliferative activity. In the three patients with singular adenoma, proliferative activity was not detected in the normal parathyroid tissue. However, in the double adenoma group, two of the three patients showed hyperproliferative activity in the normal glands. Proliferative activity consistent with hyperplasia was present in some normal glands of multiple adenoma patients. Our observation supports the theory that multiple adenomas may be a continuation of the hyperplasia state.

Evidence of Nuclear PKC/MAP-Kinase Cascade in Guinea Pig Model of Epidermal Hyperproliferation

In order to delineate the biochemical events in the nuclear compartment of an in vivo proliferating epidermis, we produced a model of hyperproliferative epidermis by topical application of docosahexaenoic acid (22:6n-3) on guinea pig skin. Employing this model we demonstrated: (i) that protein kinase C (PKC)-a and atypical PKC-zeta are the two major PKC isozymes in the normal epidermal nuclear membrane, in contrast to PKC-alpha and PKC-beta in the epidermal plasma membrane; (ii) that topical application of docosahexaenoic acid induced epidermal hyperproliferation and enhanced total nuclear PKC, particularly nuclear PKC-alpha and the atypical PKC-zeta isozymes. The increase in the nuclear PKC isozymes paralleled a marked increase in the expression of nuclear mitogen-activated protein-kinase. These data suggest that epidermal hyperproliferative activity is accompanied by the upregulation of nuclear PKC/mitogen-activated protein-kinase signaling pathway.

진주종에서 증식표지인자의 발현

Middle ear cholesteatoma is often invasive with consequent bone destruction. Inflammatory stimulation of the underlying connective tissue may be responsible for the dysrégulation and abnormal proliferative feature of the kératinocytes in cholesteatoma. Comparative investigations were performed to assess the epithelial cell kinetics of cholesteatoma and duditory meatal skin. Monoclonal antibody PCNA and Ki-67 immunohistochemical stainings were applied. Specimens of cholesteatoma samples (n = 30) showed an average PCNA score (quotient of the PCNA positive cells and the total number of cells) of 26.6 ± 10.1% and an average Ki-67 score of 15.9 ± 7.2% . Auditory meatal skin (n = 8) revealed an average PCNA score of 8.2 ± 3. 4% and an average ki-67 score of 4.9 ±0.8%. The results of this study confirm a highly increase in the proliferation rate of cholesteatoma kératinocytes, which had an PCNA score that was 3.24 times higher than the score for kératinocyte of auditory meatal skin, Ki-67 score of cholesteatoma was 3.24 times higher than auditory meatal skin. Since the cholesteatoma possesses a varible degress of proliferative activities depending on its histologic composition, the formation and accumulation of the keratin due to the continuous epithelial growth probably are important element in inducing the inflammation. The results confirm that cholesteatoma is a hyperproliferative activity and indicate that PCNA and Ki-67 immunohistochemical stainings are valuable tool for assessing cell kinetics in cholesteatoma.

Aging

Recent investigations have demonstrated a protective role for folic acid in dysplasia and neoplasia, through an unknown mechanism. The current study was designed to evaluate whether the protective role of folic acid is due, in part, to its antiproliferative properties. In addition, because colorectal neoplasia is more common with increasing age, we have compared results in both old (22 months) and young (3 months) rats. Colorectal mucosal explants of rats treated with the known carcinogen methylazoxymethanol, were supplemented with folic acid. Ornithine decarboxylase was then measured as an index of cellular proliferative activity. We observed that supplemental folic acid suppressed carcinogen-induced ornithine decarboxylase activity by 64% in the old and 74% in the young rats. Furthermore, a similar phenomenon was observed for tyrosine kinase, which was measured for comparison. The suppression of hyperproliferative activity by supplemental folic acid may contribute to the protective effect of folic acid in colorectal neoplasia.