Epithelial Cell Replication Research Articles

TCS regulator CpxR of Edwardsiella piscicida is vital for envelope integrity by regulating the new target gene yccA, stress resistance, and virulence

Edwardsiella piscicida is a serious pathogen in aquaculture and has become a model strain for the study of aquatic pathogenic bacteria. The two-component system (TCS) CpxRA is known to be intimately correlated with the adversity resistance and virulence of multiple pathogens. However, the role of CpxRA remains vague in most aquatic pathogens, especially E. piscicida. In this study, by constructing an in-frame mutant strain of cpxR, we find that CpxR of E. piscicida is closely involved in bacterial resistance to high temperature, antibiotics, and osmotic stress, which is further confirmed by the finding that CpxR is essential for cell membrane integrity. Our result also illustrates that CpxR is required for bacterial resistance against host serum and biofilm formation. Subsequent pathogenicity experiments display that CpxR is indispensable for bacterial adhesin to epithelial cell and bacterial survival and replication in phagocyte, and that CpxR is essential for bacterial colonization and dissemination in host immune tissues and general virulence. The regulatory mechanism of CpxR is clarified by the result that CpxR positively regulates the transcription of yccA, which is a new target gene of CpxR. The regulation of CpxR on yccA is decisive for envelope integrity. Taken together, these findings provide compelling evidence that CpxR of E. piscicida is not only crucial for bacterial resistance against envelope stress, but also is indispensable for bacterial virulence, which is conducive to have an insight into the importance of CpxRA system in aquatic pathogens.

NALTREXONE AS A PROMISING TREATMENT FOR CLINICAL SIGNS OF LATERAL LINE DEPIGMENTATION IN PALETTE SURGEONFISH (PARACANTHURUS HEPATUS).

Lateral line depigmentation (LLD) is a common condition in managed tropical saltwater fish, and treatment is somewhat elusive. Naltrexone, an opioid receptor antagonist, enhances epithelial cell replication, cytokine production, and angiogenesis to stimulate wound healing in mice. A treatment trial with 11 palette surgeonfish with LLD was performed. Seven fish underwent a single topical treatment of a mixture of 4 mg naltrexone and 10 g iLEX petroleum paste applied topically to LLD lesions. Four additional fish served as controls: two received only topical iLEX and two received no treatment. Severity of disease was scored on a 0-3 scale. Inflammatory response was gauged on a separate 0-3 scale for 5 d after treatment based on severity of erythema, as seen in a clinical case performed prior to this study. After 11 days, four affected animals that lacked an inflammatory response after naltrexone topical treatment were administered a single dose of intralesional 0.04% naltrexone (4 mg diluted into 10 ml saline). Lesions on all fish were photographed and measured at day 33. Clinical improvements in lesion size and pigmentation were apparent following topical naltrexone therapy in fish with severe lesions. Although these cases are promising, more data are needed to further evaluate the effectiveness of naltrexone 0.04% in treating LLD lesions in palette surgeonfish.

Review of methods for the detection of Lawsonia intracellularis infection in pigs.

Lawsonia intracellularis is an obligate intracellular bacterium associated with enteric disease in pigs. Clinical signs include weight loss, diarrhea, and, in some cases, sudden death. The hallmark lesion is the thickening of the intestinal mucosa caused by increased epithelial cell replication, known as proliferative enteropathy. The immune response to L. intracellularis is not well defined, and detection of the infection, especially in the early stages, is still a significant challenge. We review here the main approaches used to identify this important but poorly understood pathogen. Detection of L. intracellularis infection as the cause of clinical disease is confounded by the high prevalence of the pathogen in many countries and that several other pathogens can produce similar clinical signs. A single L. intracellularis–specific ELISA and several amplification assays are available commercially to aid detection and surveillance, although histopathology remains the primary way to reach a conclusive diagnosis. There are major gaps in our understanding of L. intracellularis pathogenesis, especially how the host responds to infection and the factors that drive infection toward different clinical outcomes. Knowledge of pathogenesis will increase the predictive value of antemortem tests to guide appropriate interventions, including identification and treatment of subclinically affected pigs in the early stages of disease, given that this important manifestation reduces pig productivity and contributes to the economic burden of L. intracellularis worldwide.

2062-P: Pancreatic Inflammation Implicates Increased Cellular Plasticity in the Pancreatic Duct Glands in Type 1 Diabetes

Type 1 diabetes (T1D) is an autoimmune disease characterized by endocrine/exocrine pancreas inflammation, fibrosis and, ultimately, decreased organ mass. Accumulating evidence suggests that this chronic inflammation may play a pivotal role in the initiation and progression of endogenous pancreatic cell plasticity. Pancreatic duct glands (PDGs), a potential stem/progenitor cell niche, represent a distinct anatomical compartment of the pancreas, forming blind-ending outpouchings from the main pancreatic duct and its branches. PDGs become activated in response to inflammation in T1D, evidenced by a high frequency of epithelial cell replication and an increase in progenitor-like cells. We addressed the hypothesis that in T1D, perturbation of hormone and non-hormone expressing cells in the interlobular ducts (ILD) and PDGs is a consequence of inflammation. Pancreatic sections from 21 T1D (disease duration 3 - 84 y) and 22 age-matched nondiabetic (ND) donors were evaluated for replication and expression of known pancreatic endocrine hormones in ducts and PDGs. We confirmed increased duct and PDG replication in T1D (T1D vs. ND— ILD: 2.8 ± 0.9 vs. 0.7 ± 0.1%, p<0.05; PDG: 3.4 ± 1.0 vs. 1.1 ± 0.4%, p<0.05) and reduced frequency of insulin expressing cells (T1D vs. ND— ILD: 0.0 ± 0.0 vs. 0.4 ± 0.1%, p<0.001; PDGs: 0.01 ± 0.01 vs. 0.8 ± 0.3%, p<0.05). The frequency of PDG cell replication was highest in T1D donors with acute pancreatitis. No differences were found in the frequency of other endocrine cell types regardless of the presence of pancreatic inflammation. These data suggest that increased pancreatic ductal cell replication in the PDG compartment of subjects with T1D is associated with sustained autoimmunity-related inflammation. Efforts are underway to determine specificity of pancreatic ductal cell changes to islet or acinar inflammation and the identity of replicating ILD and PDG cells, to better understand the mechanisms underlying pancreatic ductal cell plasticity. Disclosure S.S. Kulkarni: None. A.E. Butler: None. M.A. Atkinson: None.

Gremlin 1 depletion in vivo causes severe enteropathy and bone marrow failure.

The intestinal epithelium is perpetually renewed from a stem cell niche in the base of crypts to maintain a healthy bowel mucosa. Exit from this niche and maturation of epithelial cells requires tightly controlled gradients in BMP signalling, progressing from low BMP signalling at the crypt base to high signalling at the luminal surface. The BMP antagonist gremlin 1 (Grem1) is highly expressed by subepithelial myofibroblasts adjacent to the intestinal crypts but its role in regulating the stem cell niche and epithelial renewal in vivo has not been explored. To explore the effects of Grem1 loss in adulthood following normal growth and development, we bred mice (ROSA26CreER‐Grem1 flx/flx) in which Grem1 could be deleted by tamoxifen administration. While Grem1 remained intact, these mice were healthy, grew normally, and reproduced successfully. Following Grem1 depletion, the mice became unwell and were euthanised (at 7–13 days). Post‐mortem examination revealed extensive mucosal abnormalities throughout the small and large intestines with failure of epithelial cell replication and maturation, villous atrophy, and features of malabsorption. Bone marrow hypoplasia was also observed with associated early haematopoietic failure. These results demonstrate an essential homeostatic role for gremlin 1 in maintaining normal bowel epithelial function in adulthood, suggesting that abnormalities in gremlin 1 expression can contribute to enteropathies. We also identified a previously unsuspected requirement for gremlin 1 in normal haematopoiesis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract P3-06-06: Novel breast cancer mutational signatures

Abstract Background: Our understanding of the biological processes that generate somatic mutations in breast cancer has increased markedly over the past five years. Using the catalog of somatic mutations present in cancer genomes, over 30 “mutational signatures” have been produced. While these provide important insights into the processes responsible for somatic mutation, gaps remain, and the etiology of several signatures remains unknown. Methods: We have developed a new method in which the specific nucleotide change (e.g., C>T), the codon that each mutation falls in (e.g., GCT), the position in the codon (e.g., 2), and the nucleotides immediately 5' and 3' of the mutation (e.g., 5': C; 3': G) are all considered. The summary of these mutation characteristics forms a mutational profile for each tissue sample. Putting multiple samples' profiles together forms a sparse matrix with the number of samples as rows and the mutation characteristics as columns. Nonsmooth nonnegative matrix factorization was then applied to enable the discovery of intrinsic patterns in this sparse matrix. Results: Using somatic mutations identified in 1017 breast cancer tissues from The Cancer Genome Atlas (TCGA), we have identified four mutational signatures. Signature A correlates with the well-defined APOBEC signatures and signature B with the “aging” signature, which is the result 5-methylcytosine hydrolysis. Signature C and signature D are potentially new signatures. Signature D is enriched with C:G>A:T mutations; these mostly occur in the middle position of codons, and are enriched with GG(CC) either 5' or 3' of the mutation's sequence context. G>T mutations are known to occur as a consequence of oxidative damage that is not repaired. Guanines are vulnerable as they have the highest vertical oxidation potential of the nucleobases. The 5' guanines in GG sites are especially reactive. We hypothesize that Signature D results from oxidative mutagenesis. When correlated with clinical phenotypes, the basal subtype is clearly enriched for tumors with the Signature D mutation pattern (exposure level is in 169 basal tumors and in 797 non-basal tumors, p=<0.01), suggesting an etiologic link with basal-like breast cancer. G>T somatic mutations in breast cancer mainly take place during cell replication rather than during transcription. In the normal breast, epithelial cell replication occurs during the luteal phase of the menstrual cycle and during pregnancy, primarily under the direction of progesterone (P). P binds to its receptor (PR) in a subpopulation of PR positive cells where it initiates the transcription of genes including RANKL, with resultant paracrine stimulation (through RANK), of the NF-κB signaling pathway in neighboring cells. The four RANKL genes (TOP2A, MKI67, PBK, CDK1), defined by Nolan et al., are all positively associated with the signature D (P-values < 0.05), suggesting that this type of mutagenesis is associated with RANKL pathway upregulation. Conclusions: We have identified a potentially new somatic mutational signature, which we have designated as Signature D, which appears to result from exposure of DNA to oxidative stress during replication. It is associated with the basal subtype of breast cancer as well as RANKL- NF-κB pathway upregulation. Citation Format: Zeng Z, Vo AH, Luo Y, Khan SA, Clare SE. Novel breast cancer mutational signatures [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-06.

No Compensatory Increase in Non-ß Hormone Expressing Cells in the Pancreatic Duct Glands in Type 1 Diabetes

Longstanding type 1 diabetes (T1D) entails a near total loss of β cells secondary to autoimmunity, fibrosis and decreased total organ mass, but accumulating evidence suggests there may be an endogenous attempt to regenerate β cells. Pancreatic duct glands (PDGs) represent a distinct anatomical compartment of the pancreas, forming blind ending outpouchings from the main pancreatic duct and its branches. The PDG is purported to be a potential stem/progenitor cell niche. PDGs respond to inflammation in T1D by becoming activated, evidenced by a high frequency of epithelial cell replication and an increase in progenitor-like cells. Hence, we addressed the hypothesis that in T1D, perturbation of hormone-expressing endocrine cells in the interlobular ducts (ILD) and PDGs exist. Pancreatic sections from 10 T1D (disease duration 8-58 year) and 11 similarly aged control (ND) donors were evaluated for replication and expression of known pancreatic endocrine hormones in ducts and PDG. We confirmed increased duct and PDG replication in T1D (T1D vs. ND— ILD: 4.5 ± 1.8 vs. 0.8 ± 0.2%, p<0.05; PDG: 3.4 ± 1.3 vs. 0.4 ± 0.2%, p<0.05) and reduced frequency of insulin expressing cells (T1D vs. ND— ILD: 0.0 ± 0.0 vs. 0.4 ± 0.1%, p<0.001; PDGs: 0.± 0.07 vs. 2.4 ± 0.8%, p<0.05). No differences were found in the frequency of other known endocrine cell types (T1D vs. ND— ILD: Glucagon: 1.5 ± 0.7 vs. 2.1 ± 0.6%, Somatostatin: 1.2 ± 0.4 vs. 1.6 ± 0.3%, Pancreatic Polypeptide: 0.4 ± 0.2 vs. 1.3 ± 0.3%; PDGs: Glucagon: 0.9 ± 0.9 vs. 1.6 ± 1.0%, Somatostatin: 1.96 ± 0.8 vs. 3.7 ± 1.9%, Pancreatic Polypeptide: 1.7 ± 1.1 vs. 2.1 ± 0.8%; p=ns, all). These data suggest that increased cell replication and the deficiency of insulin expressing cells in the PDG compartment of subjects with T1D are not associated with a corresponding increase in non-insulin hormone-expressing endocrine cells. Furthermore, the consistent observation of increased cell replication in the ducts and PDGs in T1D may implicate regenerative mechanisms towards an exocrine cell fate. Disclosure S.S. Kulkarni: None. M.A. Atkinson: Other Relationship; Self; Patent Issued. A. Butler: None.

Murine cutaneous responses to the rocky mountain spotted fever vector, Dermacentor andersoni, feeding.

Tick salivary glands produce complex cocktails of bioactive molecules that facilitate blood feeding and pathogen transmission by modulating host hemostasis, pain/itch responses, wound healing, and both innate and adaptive immunity. In this study, cutaneous responses at Dermacentor andersoni bite-sites were analyzed using Affymetrix mouse genome arrays and histopathology at 12, 48, 96 and 120 h post- infestation (hpi) during primary infestations and 120 hpi during secondary infestations. The microarray data suggests: (1) chemotaxis of neutrophils, monocytes, and other cell types; (2) production and scavenging of reactive oxygen species; and, (3) keratin- based wound healing responses. Histological analysis supported the microarray findings. At 12 hpi, a mild inflammatory infiltrate was present in the dermis, especially concentrated at the junction between dermal connective tissue and underlying adipose tissue. A small lesion was located immediately under the hypostome and likely represents the feeding “pool.” Surprisingly, at 48 hpi, the number of inflammatory cells had not increased from 12 hpi, perhaps mirroring the reduction in gene expression seen at this time point. The feeding lesion is very well defined, and extravasated erythrocytes are readily evident around the hypostome. By 96 hpi, the inflammatory infiltrate has increased dramatically and the feeding lesion appears to have moved deeper into the dermis. At 120 hpi, most of the changes at 96 hpi are intensified. The infiltrate is very dense, the epidermis is markedly thickened, the feeding lesion is poorly defined and the dermal tissue near the hypostome appears to be loosing its normal architecture. In conclusion, during D. andersoni feeding infiltration of inflammatory cells increases across time concurrent with significant changes in the epidermal and dermal compartments near the feeding tick. The importance of changes in the epidermal layer in the host response to ticks is not known, however, it is possible the host attempts to “slough off” the tick by greatly increasing epithelial cell replication.

Histone Acetyl Transferase (HAT) HBO1 and PHD Finger Protein 17 (PHF17/JADE1) in Epithelial Cell Regeneration

HBO1 acetylates lysine residues of histones and is involved in DNA replication and gene transcription. Two isoforms of JADE1, JADE1S and JADE1L, bind HBO1 and promote acetylation of chromatinal histones. We characterized the role of JADE1‐HBO1 complexes in vitro and in vivo during epithelial cell replication. Downregulation of JADE1 by siRNA diminished the rate of DNA synthesis in cultured cells, decreased endogenous HBO1 protein expression, and prevented chromatin recruitment of replication factor Mcm7, demonstrating that JADE1 is required for cell proliferation. We used a murine model of acute kidney injury (AKI) to examine expression of HBO1‐JADE1S/L in injured and regenerating epithelial tissue. In control kidneys, JADE1S, JADE1L, and HBO1 were expressed in nuclei of proximal and distal tubular epithelial cells. Ischemia and reperfusion injury resulted in an initial decrease in JADE1S, JADE1L, and HBO1 protein levels which returned to the baseline during renal recovery. HBO1 and JADE1S recovered as cell proliferation reached its maximum while JADE1L recovered after bulk proliferation had ceased. The temporal expression of JADE1S correlated with the acetylation of histone H4 on lysine 5 and 12, but not with acetylation of histone H3 on lysine 14, demonstrating that the JADE1S‐HBO1 complex specifically marks H4 during epithelial cell proliferation. These data implicated JADE1‐HBO1 complex in AKI and suggested distinct roles for JADE1 isoforms during epithelial cells recovery.NIH RO1 DK087910, and ACS IRG‐72–001‐32 (to M. V. P.). NIH 1KO8DK090143 (to A.H.).

Histone Acetyl Transferase (HAT) HBO1 and JADE1 in Epithelial Cell Regeneration

HBO1 acetylates lysine residues of histones and is involved in DNA replication and gene transcription. Two isoforms of JADE1, JADE1S and JADE1L, bind HBO1 and promote acetylation of histones in chromatin context. We characterized the role of JADE1-HBO1 complexes invitro and invivo during epithelial cell replication. Down-regulation of JADE1 by siRNA diminished the rate of DNA synthesis in cultured cells, decreased endogenous HBO1 protein expression, and prevented chromatin recruitment of replication factor Mcm7, demonstrating that JADE1 is required for cell proliferation. We used a murine model of acute kidney injury to examine expression of HBO1-JADE1S/L in injured and regenerating epithelial tissue. In control kidneys, JADE1S, JADE1L, and HBO1 were expressed in nuclei of proximal and distal tubular epithelial cells. Ischemia and reperfusion injury resulted in an initial decrease in JADE1S, JADE1L, and HBO1 protein levels, which returned to baseline during renal recovery. HBO1 and JADE1S recovered as cell proliferation reached its maximum, whereas JADE1L recovered after bulk proliferation had ceased. The temporal expression of JADE1S correlated with the acetylation of histone H4 on lysines 5 and 12, but not with acetylation of histone H3 on lysine 14, demonstrating that the JADE1S-HBO1 complex specifically marks H4 during epithelial cell proliferation. These data implicate JADE1-HBO1 complex in acute kidney injury and suggest distinct roles for JADE1 isoforms during epithelial cell recovery.

Insulin-like growth factor and fibroblast growth factor expression profiles in growth-restricted fetal sheep pancreas

Placental insufficiency results in intrauterine growth restriction (IUGR), impaired fetal insulin secretion and less fetal pancreatic β-cell mass, partly due to lower β-cell proliferation rates. Insulin-like growth factors (IGFs) and fibroblast growth factors (FGFs) regulate fetal β-cell proliferation and pancreas development, along with transcription factors, such as pancreatic and duodenal homeobox 1 (PDX-1). We determined expression levels for these growth factors, their receptors and IGF binding proteins in ovine fetal pancreas and isolated islets. In the IUGR pancreas, relative mRNA expression levels of IGF-I, PDX-1, FGF7 and FGFR2IIIb were 64% (P < 0.01), 76% (P < 0.05), 76% (P < 0.05) and 52% (P < 0.01) lower, respectively, compared with control fetuses. Conversely, insulin-like growth factor binding protein 2 (IGFBP-2) mRNA and protein concentrations were 2.25- and 1.2-fold greater (P < 0.05) in the IUGR pancreas compared with controls. In isolated islets from IUGR fetuses, IGF-II and IGFBP-2 mRNA concentrations were 1.5- and 3.7-fold greater (P < 0.05), and insulin mRNA was 56% less (P < 0.05) than control islets. The growth factor expression profiles for IGF and FGF signaling pathways indicate that declines in β-cell mass are due to decreased growth factor signals for both pancreatic progenitor epithelial cell and mature β-cell replication.

Latent Membrane Protein 1 Is Dispensable for Epstein-Barr Virus Replication in Human Embryonic Kidney 293 cells

Epstein Barr Virus (EBV) replicates in oral epithelial cells and gains entry to B-lymphocytes. In B-lymphocytes, EBV expresses a restricted subset of genes, the Latency III program, which converts B-lymphocytes to proliferating lymphoblasts. Latent Membrane Protein 1 (LMP1) and the other Latency III associated proteins are also expressed during virus replication. LMP1 is essential for virus replication and egress from Akata Burkitt Lymphoma cells, but a role in epithelial cell replication has not been established. Therefore, we have investigated whether LMP1 enhances EBV replication and egress from HEK293 cells, a model epithelial cell line used for EBV recombinant molecular genetics. We compared wild type (wt) and LMP1-deleted (LMP1Δ) EBV bacterial artificial chromosome (BAC) based virus replication and egress from HEK293. Following EBV immediate early Zta protein induction of EBV replication in HEK293 cells, similar levels of EBV proteins were expressed in wt- and LMP1Δ-infected HEK293 cells. LMP1 deletion did not impair EBV replication associated DNA replication, DNA encapsidation, or mature virus release. Indeed, virus from LMP1Δ-infected HEK293 cells was as infectious as EBV from wt EBV infected HEK cells. Trans-complementation with LMP1 reduced Rta expression and subsequent virus production. These data indicate that LMP1 is not required for EBV replication and egress from HEK293 cells.

Abnormal expression of telomerase/apoptosis limits type II alveolar epithelial cell replication in the early remodeling of usual interstitial pneumonia/idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis is a distinctive, usually fatal, type of chronic fibrosing interstitial pneumonia of unknown cause that increases in prevalence with advanced age, characterized by failure of alveolar re-epithelization and progressive scar formation. Recently, limitation of the replicative capacity of tissues determined by telomerase/apoptosis balance has been implicated in pathogenesis of age-related diseases. In this study, we validated the importance of the expression of type 2 alveolar epithelial cells telomerase protein and studied the relationships between telomerase and apoptosis in early remodeling of usual interstitial pneumonia. We determined type 2 alveolar epithelial cells density, telomerase expression, and apoptosis in surgical lung biopsies from 24 patients with usual interstitial pneumonia, and in normal lung tissues from 18 subjects. We used immunohistochemistry, deoxynucleotidyl transferase method of end labeling, electron microscopy, and histomorphometry to evaluate the amount of type 2 alveolar epithelial cells staining for surfactant-A, telomerase, and in situ detection of apoptotic cells. Unaffected areas of usual interstitial pneumonia and normal lung tissue had similar densities of type 2 alveolar epithelial cells, but a significant minor subpopulation of type 2 alveolar epithelial cells was telomerase positive and a large population was telomerase negative. A significant inverse association was found between low type 2 alveolar epithelial cell telomerase expression and high apoptosis in unaffected areas of usual interstitial pneumonia. Although type 2 alveolar epithelial cell telomerase expression was higher than apoptosis in NLT group, no significant association was found between them. Electron microscopy confirmed epithelial apoptosis, alveolar collapse, and initial fibroplasia. We conclude that abnormal type 2 alveolar epithelial cells telomerase/apoptosis balance may reduce alveolar epithelial regenerative capacity, thus contributing to the early remodeling response in usual interstitial pneumonia.

Induction of Epithelial Mesenchimal Transition and Vasculogenesis in the Lenses of Dbl Oncogene Transgenic Mice

BackgroundThe Dbl family of proteins represents a large group of proto-oncogenes involved in cell growth regulation. The numerous domains that are present in many Dbl family proteins suggest that they act to integrate multiple inputs in complicated signaling networks involving the Rho GTPases. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders and neoplastic transformation. We generated transgenic mice introducing the cDNA of Dbl oncogene linked to the metallothionein promoter into the germ line of FVB mice and found that onco-Dbl expression in mouse lenses affected proliferation, migration and differentiation of lens epithelial cells.ResultsWe used high density oligonucleotide microarray to define the transcriptional profile induced by Dbl in the lenses of 2 days, 2 weeks, and 6 weeks old transgenic mice. We observed modulation of genes encoding proteins promoting epithelial-mesenchymal transition (EMT), such as down-regulation of epithelial cell markers and up-regulation of fibroblast markers. Genes encoding proteins involved in the positive regulation of apoptosis were markedly down regulated while anti-apoptotic genes were strongly up-regulated. Finally, several genes encoding proteins involved in the process of angiogenesis were up-regulated. These observations were validated by histological and immunohistochemical examination of the transgenic lenses where vascularization can be readily observed.ConclusionOnco-Dbl expression in mouse lens correlated with modulation of genes involved in the regulation of EMT, apoptosis and vasculogenesis leading to disruption of the lens architecture, epithelial cell proliferation, and aberrant angiogenesis. We conclude that onco-Dbl has a potentially important, previously unreported, capacity to dramatically alter epithelial cell migration, replication, polarization and differentiation and to induce vascularization of an epithelial tissue.

Colorectal carcinogenesis: Review of human and experimental animal studies

This review gives a comprehensive overview of cancer development and links it to the current understanding of tumorigenesis and malignant progression in colorectal cancer. The focus is on human and murine colorectal carcinogenesis and the histogenesis of this malignant disorder. A summary of a model of colitis-associated colon tumorigenesis (an AOM/DSS model) will also be presented. The earliest phases of colorectal oncogenesis occur in the normal mucosa, with a disorder of cell replication. The large majority of colorectal malignancies develop from an adenomatous polyp (adenoma). These can be defined as well-demarcated masses of epithelial dysplasia, with uncontrolled crypt cell proliferation. When neoplastic cells pass through the muscularis mucosa and infiltrate the submucosa, they are malignant. Carcinomas usually originate from pre-existing adenomas, but this does not imply that all polyps undergo malignant changes and does not exclude de novo oncogenesis. Besides adenomas, there are other types of pre-neoplasia, which include hyperplastic polyps, serrated adenomas, flat adenomas and dysplasia that occurs in the inflamed colon in associated with inflammatory bowel disease. Colorectal neoplasms cover a wide range of pre-malignant and malignant lesions, many of which can easily be removed during endoscopy if they are small. Colorectal neoplasms and/or pre-neoplasms can be prevented by interfering with the various steps of oncogenesis, which begins with uncontrolled epithelial cell replication, continues with the formation of adenomas and eventually evolves into malignancy. The knowledge described herein will help to reduce and prevent this malignancy, which is one of the most frequent neoplasms in some Western and developed countries.

Resveratrol down-regulates the growth and telomerase activity of breast cancer cells in vitro

A number of previous studies investigated the in vitro effects of resveratrol on malignant human breast epithelial cell replication. The aim of the present study was to evaluate the activity of resveratrol on human metastatic breast cancer cells. The study was performed on the MCF-7 tumor cell line. Cell growth, cell cycle perturbation and apoptosis were evaluated by trypan blue dye exclusion assay, flow cytometric analysis and confocal fluorescence microscopy. TRAP assay and Western blot analysis respectively detected levels of telomerase activity and levels of hTERT in intracellular compartments of MCF-7 cells treated with resveratrol. Resveratrol has a direct inhibitory effect on cell proliferation. The results demonstrate that the drug induces apoptosis in MCF-7 cells, in a time- and concentration-related manner. Our results also show that the growth-inhibitory effect of resveratrol on malignant cells is mainly due to its ability to induce S-phase arrest and apoptosis in association with reduced levels of telomerase activity. In particular, TRAP assay and Western blot analysis respectively showed that resveratrol treatment down-regulates the telomerase activity of target cells and the nuclear levels of hTERT, the reverse transcriptase subunit of the telomerase complex. In our experimental model of breast cancer, resveratrol shows direct antiproliferative and pro-apoptotic effects. Studies on telomerase function and intracellular hTERT distribution point out that this agent is endowed with additional suppressive functions on critical tumor biological properties. These results speak in favor of a potential role of resveratrol in chemoprevention/chemotherapy of breast cancer.

Mitogen-Activated Protein Kinase Inhibitor, PD98059, Inhibits Rat Retinal Pigment Epithelial Cell Replication by Cell Cycle Arrest

Purpose: To investigate the effect of PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, on the replication of rat cultured retinal pigment epithelial (RPE) cells. Methods: Growth-phase rat RPE cells were exposed to various concentrations of PD98059 in serum-free F12 medium containing 0.1% dimethyl sulfoxide. Cell proliferation was assessed by cell counts using a hemocytometer. Cell viability was tested by CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay at 24 hours after PD98059 application. Hoechst 33552 and propidium iodide staining were used to assess nuclear morphology. Immunostaining with Ki67 antibody was used for cell cycle analysis because the staining patterns produced on cells are characteristic depending on their position within the cell cycle. Results: PD98059 inhibited cellular proliferation of cultured rat RPE cells in a dose-dependent manner but did not induce cell death. Twenty-four hours after the application of PD98059, cultured RPE cells were not immunopositive for Ki67, indicating that their cell cycle was arrested in the G0/G1 phase. Conclusion: These results demonstrated that MAPK inhibition arrested cell cycle progression of rat cultured RPE cells at the G0/G1 phase. The pharmacological induction of cell cycle arrest could be a new approach to inhibit cellular proliferation in such conditions as proliferative vitreoretinopathy.

A Double-Label Immunohistochemical Method for Assessment of Epithelial Cell Replication in Paraffin Sections of Toxicant-Damaged Lungs

Enhanced epithelial cell replication is a sensitive indicator of toxicant-induced lung damage. Cell identification in the deep lung can be difficult and limit the usefulness of conventional techniques for examining cell-cycle kinetics in toxicant-damaged lungs. The purpose of this study was to develop a technique to reliably differentiate epithelial from nonepithelial cells in the deep lung. Formalin fixed, paraffin sections of toxicant-exposed lungs were used to develop a double, sequential, peroxidase/alkaline phosphatase method to detect bromodeoxyuridine and cytokewtins. Use of lü-5 cytokeratin antibody, which binds to all cytokeratin polypeptides and is epithelial cell specific, and a biotinstreptavidin amplified detection system maximized the detection of cytokeratin immunoreactivity and allowed cells with a negative reaction to be classified as nonepithelial. The staining produced was distinct and readily detected by light microscopy. In foci of severe damage, the cytokeratin immunostaining of epithelial cells highlighted the general architecture of the parenchyma. Cell counts and labeling data from inflammatory foci in endotoxin-instilled rat lungs were compared using the double-label versus a conventional single-label immunohistochemical method. The proportion of cells that could be identified was significantly increased in the double-label slides. We conclude that in lungs with severe damage, where the usual morphologic features such as cell size, shape, vacuolization, and position are unreliable indicators of cell type, the double-label procedure would aid the microscopist in obtaining reliable cells counts. (The J Histotechnol 25:15, 2002)Submitted: September 10, 2001; Accepted with revisions: November 7, 2001

A Double-Label Immunohistochemical Method for Assessment of Epithelial Cell Replication in Paraffin Sections of Toxicant-Damaged Lungs

A Double-Label Immunohistochemical Method for Assessment of Epithelial Cell Replication in Paraffin Sections of Toxicant-Damaged Lungs

Pathology of colorectal cancer.

The earliest phases of colorectal tumourigenesis initiate in the normal mucosa, with a generalised disorder of cell replication, and with the appearance of clusters of enlarged crypts (aberrant crypts) showing proliferative, biochemical and biomolecular abnormalities. The large majority of colorectal malignancies develop from adenomatous polyps. These can be defined as well demarcated masses of epithelial dysplasia, with uncontrolled crypt cell division. An adenoma can be considered malignant when neoplastic cells pass through the muscularis mucosae and infiltrate the submucosa. Definitions like "carcinoma in situ" or "intramucosal carcinoma" should be abandoned, since they lead to confusion. Although several lines of evidence indicate that carcinomas usually originate from pre-existing adenomas, this does not imply that all polyps undergo malignant changes, and does not exclude "de novo" carcinogenesis. Besides adenomas, other types of polypoid lesions include hyperplastic polyps (showing elongated crypts often with cystic dilatation), serrated adenomas (with a serrated glandular pattern], flat adenomas (flat lesions which are difficult to detect in routine lower endoscopy, but may possess malignant potential), hamartomatous polyps (which show a complex branching pattern of smooth muscle supporting normal lamina propria and glands), and inflammatory polyps. Colorectal carcinomas are one of the most frequent neoplasms in Western society; the macroscopic appearance of these lesions may be that of a polypoid vegetating mass or of a flat infiltrating lesion. Most of these tumours are adenocarcinomas (96%), that, in some cases, show a mucinous component. More rare malignancies of the large bowel include signet-ring cell carcinoma, squamous carcinoma, undifferentiated neoplasms and medullary type adenocarcinoma (solid carcinoma with minimal glandular differentiation or slight cellular pleomorphism). Colorectal carcinoma can be graded into well, moderately and poorly differentiated lesions; there is little evidence, however, that grading may be of help in evaluating prognosis of affected patients. In conclusion, colorectal tumours cover a wide range of premalignant and malignant lesions, many of which can easily be removed at endoscopy. It follows that colorectal neoplasms might be prevented by interfering with the various steps of carcinogenesis, which begins with uncontrolled epithelial cell replication, continues with the formation of adenomas of various dimensions, and eventually evolves into malignancy.