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Background/aim Chronic inflammation generates large quantities of reactive oxygen and nitrogen species that damage DNA. DNA repair is important for cellular viability and genome integrity. Materials and methods Expression levels of the DNA repair proteins OGG1, XPA, MLH1, PARP1, and XRCC6, which function in base excision repair, nucleotide excision repair, mismatch repair, single-strand break repair and double-strand break repair, respectively, were assessed using immunohistochemistry in ulcerative colitis and sporadic colorectal cancer biopsies. Levels of oxidative/ nitrosative stress biomarkers were also assessed. Results Ulcerative colitis and colorectal cancer lesions expressed significantly higher levels of all DNA repair proteins and oxidative/ nitrosative stress biomarkers compared to normal colonic mucosa. Ulcerative colitis had the highest XPA and XRCC6 expression. Conclusion Oxidative/nitrosative stress is prevalent in the colon of both diseases. Nucleotide excision repair and non-homologous end-joining double-strand break repair may be compromised in colorectal cancer, but not in ulcerative colitis.

Background Keratoacanthoma (KA) has a unique life cycle of rapid growth and spontaneous regression that shows similarities to the hair follicle cycle, which involves an active Wnt signaling during physiological regeneration. We analyzed the expression of the Wnt signaling proteins β-catenin, Lef1, Sox9, and Cyclin D1 in young and old human KAs to investigate a possible role for Wnt signaling in KAs. Aim To investigate the role of the Wnt/β-catenin signaling pathway in human KAs. Methods and results Formalin-fixed, paraffin-embedded tissue samples of 67 KAs were analyzed for protein expression using immunohistochemistry. The majority of KAs were positive for Sox9 and Cyclin D1 but not for nuclear-localized β-catenin or Lef-1. No significant differences in protein expressions were seen between young and old KAs. However, we found a significant association between Ki67 and Cyclin D1 proteins (P= .008). Conclusions The Wnt signaling pathway does not appear to play a significant role in the biogenesis of human KA. Sox9 overexpression may be indicative of inhibition of Wnt signaling. Sox-9 and Cyclin D1 are proliferation markers that are most likely transactivated by alternate signaling pathways.

Background Keratoacanthoma (KA) is a common keratinocytic skin neoplasm that typically develops rapidly and undergoes complete spontaneous regression. As the pro-apoptotic p53 protein may be involved in the lifecycle of KA, we studied the p53 status throughout the main stages of KA that include proliferation, maturation and regression in a large series of lesions. Methods One-hundred and twenty-four KAs were characterized with respect to age of the lesions both clinically and histopathologically, in addition to phenotypic characteristics such as cellular atypia, infiltration, inflammation and fibrosis. Tp53 mutations were detected by capillary electrophoresis, and p53 protein levels were assessed by immunohistochemistry. Results Tp53 mutations were detected in 49 cases (39.5%) and were associated with high p53 protein levels (p = 0.007) and histopathologic age of the lesions (p = 0.044). Significant association was also seen between high p53 protein levels and atypia (p = 0.036), whereas the association with infiltration showed borderline significance (p = 0.057). High p53 protein levels were significantly associated with gene mutations in transplanted, but not in non-transplanted patients. Conclusion We show a high frequency of Tp53 mutations in KAs that is associated with increased p53 levels. The results indicate a role for the p53 protein in KA development.

Background The colon and rectum are continuously exposed to oxidative stress that generates reactive oxygen species, which are a major cause of DNA double-strand breaks (DSB). Furthermore, chronic inflammatory diseases such as ulcerative colitis (UC) are characterized by an excess of reactive nitrogen species that can also lead to DNA double-strand breakage and genomic instability. We investigated the expression of the nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1) protein in UC and sporadic colorectal cancer (CRC) due to its involvement in both DNA double-strand break repair and inflammatory signaling. Methods NUCKS1 expression and expression of the DNA double-strand break marker gamma-H2AX (γH2AX) were assessed in formalin-fixed, paraffin-embedded UC and CRC patient biopsies using peroxidase immunohistochemistry. Expression levels for both proteins were evaluated together with previously published expression-level data for hTERT and TP53 proteins in the same material. Results Nondysplastic UC lesions had 10-fold lower γH2AX expression and approximately 4-fold higher NUCKS1 expression compared with sporadic CRC, indicating minimal DNA DSB damage and heightened DNA DSB repair in these lesions, respectively. NUCKS1 expression in UC tended to decrease with increasing grades of dysplasia, whereas γH2AX, hTERT, and TP53 expression tended to increase with increasing grades of dysplasia. The highest γH2AX expression was seen in sporadic CRC, indicating considerable DNA DSB damage, whereas the highest NUCKS1 expression and hTERT expression were seen in nondysplastic UC. Conclusions Overall, our data suggest that NUCKS1 may be involved in DNA DSB repair and/or inflammatory signaling in UC, but a more thorough investigation of both pathways in UC is warranted.

Aneuploidy is a common feature in the colonic mucosa of patients suffering from the inflammatory bowel disease ulcerative colitis (UC) and often precedes the development of dysplasia and cancer. Aneuploidy is assumed to be caused by missegregation of chromosomes during mitosis, often due to a faulty spindle assembly checkpoint. p53 is a tumour suppressor protein known to regulate the spindle assembly checkpoint and is frequently mutated in aneuploid cells. Aurora A is a presumed oncoprotein, also involved in regulation of the spindle assembly checkpoint. In the present study, we examined the mutational frequency of TP53 and the protein levels of p53 in a set of 20 progressor and 10 non-progressor colectomies from patients suffering from longstanding UC. In addition, we re-examined previously published immunohistochemical data on Aurora A expression using the same material. Levels of Aurora A were re-examined with regard to DNA ploidy status and dysplasia within the progressors, as well as in relation to p53 accumulation and TP53 mutational status. We detected p53 accumulation only within the progressor colectomies, where it could be followed back 14 years prior to the colectomies, in pre-colectomy biopsies. TP53 mutations were detected in both progressors and non-progressors. Expression levels of Aurora A were similar in the progressors and non-progressors. Within the group of progressors however, low levels of Aurora A were associated with areas of DNA aneuploidy, as well as with increasing degrees of dysplasia. Our results indicate that alterations in p53 may be an early biomarker of a progressor colon, and that p53 is accumulated early in UC-related carcinogenesis. Furthermore, a decreased Aurora A expression is associated with the development of DNA aneuploidy, as well as with dysplasia in UC progressors.

Longstanding ulcerative colitis (UC) is a disease of chronic inflammation of the colon. It is associated with the development of colorectal cancer through a multistep process including increasing degrees of dysplasia and DNA-ploidy changes. However, not all UC patients will develop these characteristics even during lifelong disease, and patients may therefore be divided into progressors who develop dysplasia or cancer, and non-progressors who do not exhibit such changes. In the present study, the amount of hTERT, the catalytic subunit of the enzyme telomerase, was estimated by using peroxidase immunohistochemistry (IHC) in a set of progressor and non-progressor UC colectomies. The protein levels in the colonic mucosa of the progressors and non-progressors were compared, and further comparisons between different categories of dysplastic development and to DNA-ploidy status within the progressors were made. Levels of hTERT were elevated in the colonic mucosa of the progressors and non-progressors when compared to non-UC control samples, but no difference was observed between the hTERT levels in the mucosa of progressors and non-progressors. The levels of hTERT associated with levels of Ki67 to a significant degree within the non-progressors. hTERT expression in lesions with DNA-aneuploidy were decreased as compared to diploid lesions, when stratified for different classes of colonic morphology. Our results indicate an association between hTERT protein expression and aneuploidy in UC-progressor colons, and also a possible protective mechanism in the association between hTERT and Ki67, against development of malignant features within the mucosa of a UC-colon.

Intratumor heterogeneity in chromosomal aberrations is believed to represent a major challenge in the treatment of cancer. The aim of our work was to assess the chromosomal heterogeneity of advanced cervical carcinomas and to distinguish aberrations that had occurred at a late stage of the disease from early events. A total of 55 biopsies, sampled from 2–4 different sites within 20 tumors, were analyzed by use of comparative genomic hybridization. Heterogeneous aberrations were identified as those present in at least 1 of the biopsies and which were not seen, nor seen as a tendency, in the others of the same tumor. The homogeneous aberrations were those seen in all biopsies of the tumor. The most frequent homogeneous aberrations were gain of 3q (65%), 20q (65%) and 5p (50%), indicating that these are early events in the development of the disease. Chromosomal heterogeneity was observed in 11 tumors. The most frequent heterogeneous aberrations were loss of 4p14–q25 (60% of 10 cases with this aberration), and gain of 2p22–pter (50% of 6 cases), 11qcen–q13 (33% of 9 cases) and 8q (27% of 11 cases), suggesting that these events promote progression at a later stage. Many of the heterogeneous regions contained genes known to influence the prognosis of cervical cancer, such as 7p (EGFR), 8q (c-MYC), 11qcen-q13 (CCND1) and 17q (ERBB2). Three evolution sequences for the subpopulations in the heterogeneous tumors were identified: a serial, a parallel and a mixed sequence. In 2 tumors with a serial sequence, it was indicated that the aberrations +8 and −X had occurred after the other heterogeneous aberrations and hence were the aberrations most recently formed. Our results suggest pronounced chromosomal instability in advanced cervical carcinomas. Moreover, aggressive and treatment-resistant subpopulations may emerge at a late stage and possibly contribute to a poor prognosis of the advanced stages. © 2004 Wiley-Liss, Inc.

SUMMARYWe show that crucian carp (Carassius carassius) living in normoxic(aerated) water have gills that lack protruding lamellae, the primary site of O2 uptake in fish. Such an unusual trait leads to a very small respiratory surface area. Histological examination showed that the lamellae(secondary lamellae) of these fish were embedded in a cell mass (denoted embedded lamellae). When the fish were kept in hypoxic water, a large reduction in this cell mass occurred, making the lamellae protrude and increasing the respiratory surface area by ∼7.5-fold. This morphological change was found to be reversible and was caused by increased apoptosis combined with reduced cell proliferation. Carp with protruding lamellae had a higher capacity for oxygen uptake at low oxygen levels than fish with embedded lamellae, but water and ion fluxes appeared to be increased, which indicates increased osmoregulatory costs. This is, to our knowledge, the first demonstration of an adaptive and reversible gross morphological change in the respiratory organ of an adult vertebrate in response to changes in the availability of oxygen.

Background Ulcerative colitis (UC) is a chronic, inflammatory bowel disease which may lead to dysplasia and adenocarcinoma in patients when long-lasting. Short telomeres have been reported in mucosal cells of UC patients. Telomeres are repetitive base sequences capping the ends of linear chromosomes, and protect them from erosion and subsequent wrongful recombination and end-to-end joining during cell division. Short telomeres are associated with the development of chromosomal instability and aneuploidy, the latter being risk factors for development of dysplasia and cancer. Specifically, the abrupt shortening of one or more telomeres to a critical length, rather than bulk shortening of telomeres, seems to be associated with chromosomal instability. Methods We investigated possible associations between dysplasia, aneuploidy and telomere status in a total of eight lesions from each of ten progressors and four nonprogressors suffering from longstanding UC. We have analyzed mean telomere length by qPCR, as well as the amount of ultra-short telomeres by the Universal STELA method. Results An increased amount of ultra-short telomeres, as well as general shortening of mean telomere length are significantly associated with dysplasia in longstanding UC. Furthermore, levels of ultra-short telomeres are also significantly increased in progressors (colons harbouring cancer/dysplasia and/or aneuploidy) compared to nonprogressors (without cancer/dysplasia/aneuploidy), whereas general shortening of telomeres did not show such associations. Conclusions Our data suggest that ultra-short telomeres may be more tightly linked to colorectal carcinogenesis through development of dysplasia in UC than general telomere shortening. Telomere status was not seen to associate with DNA aneuploidy.

BACKGROUND Bax, Bcl-2, and p53 proteins are involved in the regulation of apoptosis and have been reported to correlate with prognosis in several tumor types. METHODS Bax, Bcl-2, p53, and the level of spontaneous apoptosis were evaluated in formalin fixed, paraffin embedded pretreatment specimens from 85 T1–4 squamous cell carcinomas (SCCs) of the tongue by immunohistochemical methods. The percentage of apoptotic cells labeled by the terminal deoxynucleotidyl transferase (TdT)–mediated dUTP labeling (TUNEL) method was expressed as an apoptotic index (AI). For Bax and Bcl-2 evaluation, the fraction of tumor cells stained and the staining intensities were given scores that were added together, resulting in a final score. p53 immunostaining was expressed as a percentage of positive cells. RESULTS High AI was significantly associated with high Bax expression (P = 0.0122) and highly differentiated tumors (P = 0.0062). No correlation was found between AI and Bcl-2 expression. There was no correlation between p53 positivity and any of the other apoptosis-related parameters. Whereas low AI scores and low Bax expression correlated significantly with poor prognosis (P = 0.0053 and P = 0.0012, respectively), a low Bcl-2 expression was associated with a favorable clinical outcome (P = 0.0262). Patients with a high Bcl-2/Bax expression ratio had a significantly poorer prognosis than those with a low ratio (P < 0.0001). Multivariate analysis revealed that Bax expression, the Bcl-2/Bax expression ratio, and the T and N classifications were significantly independent prognostic variables. The Bcl-2/Bax expression ratio was the strongest independent prognostic parameter. CONCLUSIONS AI, individual Bax and Bcl-2 expression, and particularly the Bcl-2/Bax expression ratio have prognostic value in SCC of the tongue. Cancer 1999;86:913–20. © 1999 American Cancer Society.

The hypothesis to be tested was that abnormal sperm chromatin structure is related to disturbed spermatogenesis in patients with testicular cancer. After orchiectomy but before further treatment ("pretreatment"), semen samples from 39 patients with testicular cancer were analyzed for sperm concentration by light microscopy and by the sperm chromatin structure assay (SCSA). In 28 patients assessment of sperm concentration was repeated 12-26 months after orchiectomy ("posttreatment"). The pretreatment SCSA results for the patients were compared to those from 18 healthy semen donors and assessed for correlation with the patients' posttreatment sperm concentration. Twenty-three patients displayed an abnormal chromatin structure in their pretreatment sample. For the nine evaluable patients on the surveillance program, the pretreatment SCSA results were not correlated with the posttreatment concentration. The results from 19 evaluable patients undergoing cytotoxic treatment (radiotherapy, 13; chemotherapy, 6) indicate that posttreatment recovery of spermatogenesis (recovery in 4 of 5 patients) is observed more often in patients with a normal pretreatment chromatin structure than in those with abnormal SCSA values before treatment (recovery in 2 of 14 patients; P = 0.02). The results of SCSA display sperm characteristics beyond those of light microscopically assessed sperm concentration. Pretreatment SCSA results might help clinicians to identify those testicular cancer patients with a high risk of long-lasting posttreatment disturbance of spermatogenesis.

The sperm chromatin structure assay (SCSA) measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ, and was developed on two Ortho flow cytometers, an FC200 and a cytofluorograf 30 (BDIS), both having orthogonal axes of fluorochrome excitation, emission, and sample flow. Sperm cells are first treated with a pH 1.4 buffer to denature DNA in situ and then stained with the metachromatic dye acridine orange (AO). The metachromatic fluorescence measured reflects relative amounts of denatured (red fluorescence) and native (green fluorescence) DNA present per cell. The extent of DNA denaturation is quantified by the calculated parameter alpha t [{alpha}{sub t} = red/(red + green) fluorescence]. Alpha t variables important for correlations with fertility and toxicant-induced chromatin damage include mean (X{alpha}{sub t}), standard deviation (SD{alpha}{sub t}), and cells outside the main population (COMP{alpha}{sub t}). This study showed that the SCSA can be successfully run on two epiillumination-type instruments, an Ortho ICP22A and Skatron Argus {trademark}, and two additional orthogonal axes instruments, a Becton Dickinson FACScan {trademark} and a Coulter Elite {trademark}. Epiillumination instruments produced a different fluorescence distribution than orthogonal instruments, but the resulting {alpha}{sub t} values showed strong conformity and interpretation of results was the same. SCSAmore » values obtained on the Coultier Elite {trademark} were most similar to the Cytofluorograf 30; the FACScan {trademark} green fluorescence distribution was narrower and allowed resolution of cell doublets. Neither orthogonal instrument has the ability to directly calculate {alpha}{sub t} values. Listmode data from these instruments were transferred to an off-line personal computer (PC) for calculation of {alpha}{sub t} values using LIST-VIEW {trademark} software. 28 refs., 5 figs., 2 tabs.« less

Data are limited on oncogene mutation frequencies in polyps from principally asymptomatic participants of population-based colorectal cancer screening studies. In this study, DNA from 204 polyps, 5 mm or larger, were collected from 176 participants of the NORCCAP screening study and analyzed for mutations in KRAS, BRAF, and PIK3CA including the rarely studied KRAS exons 3 and 4 mutations. KRAS mutations were identified in 23.0% of the lesions and were significantly associated with tubulovillous adenomas and large size. A significantly higher frequency of KRAS mutations in females was associated with mutations in codon 12. The KRAS exon 3 and 4 mutations constituted 23.4% of the KRAS positive lesions, which is a larger proportion compared to previous observations in colorectal cancer. BRAF mutations were identified in 11.3% and were associated with serrated polyps. None of the individuals were diagnosed with de novo or recurrent colorectal cancer during the follow-up time (median 11.2 years). Revealing differences in mutation-spectra according to gender and stages in tumorigenesis might be important for optimal use of oncogenes as therapeutic targets and biomarkers.

We studied patterns of DNA damage signaling and cell cycle response to clinically-relevant (bolus) and high doses of 5-fluorouracil (5-FU) in three colorectal cancer cell lines with differing MMR and TP53 status in an attempt to better understand how 5-FU exerts its cytotoxicity. The ATM/CHEK2/ CHEK1 signaling pathway was not activated in response to bolus 5-FU in the MMR-deficient cell lines HCT116 (TP53- proficient or TP53-depleted) and HCT15 (TP53-deficient), consistent with negligible/reparable DNA damage and no cell death. The pattern of DNA damage checkpoint activation in bolus 5-FU-treated HT29 (TP53-deficient/MMR-proficient) cultures suggested SSB formation (CHEK1 activation) followed by DSB formation (CHEK2 activation and increased phospho- H2AX levels), but no cell death suggested that DNA repair capacity was not overwhelmed. High-dose 5-FU treatment led to activation of ATM/CHEK2/TP53 (not CHEK1) in TP53- proficient and TP53-depleted HCT116 (later CHEK2 activation relative to TP53-proficient) cultures; HCT15 cultures had ATM activation only. These data and increased phospho-H2AX levels indicated DSB formation; apoptosis was induced in both cell lines indicating irreparable DNA damage. TP53-depleted HCT116 cultures also had DSBs after high-dose 5-FU treatment but experienced a (transient) G1/S cell cycle arrest that protected them from apoptosis. TP53 phosphorylation at Ser20/33/37 was seen in TP53-proficient HCT116 cultures regardless of 5-FU concentration at ≥4 h following treatment, indicating TP53 stabilization/transcriptional activation. Overall, activation of ATM, CHEK1 and/or CHEK2 and phospho-H2AX levels reflected the nature of 5-FU-induced DNA damage and indi - cated when DNA damage was signi ficant (5-FU-dose-dependent). DNA repair and cell cycle responses to 5-FU-induced DNA damage were distinctly affected by MMR and TP53 (role in BER/NER) functionalities, but MMR deficiency especially seemed to confer less overall sensitivity to 5-FU.

Cell cycle checkpoints ensure that eukaryotic cells do not enter mitosis after ionizing irradiation (IR). The G(2)-arrest after IR is the result of activation of multiple signalling pathways, the contributions of which vary with time after irradiation. We have studied the time evolution of the IR-induced G(2)-arrest in human B-lymphocyte cancer cell lines, as well as the molecular mechanisms responsible for the arrest. Cells that were in G(2) phase at the time of irradiation experienced a transient arrest that blocked entry into mitosis at 0-2 hours after IR (0.5 or 4 Gy). Activation of ATM and CHEK2 occurred at the same time as this early arrest and was, like the arrest, abrogated by the ATM-inhibitor KU-55933. A late, permanent and ATM-independent arrest (≥6 hours after IR) of cells that were in G(2)/S/G(1) at the time of irradiation (4 Gy) was inactivated by caffeine. This late G(2)-arrest could not be explained by down-regulation of genes with functions in G(2)/mitosis (e.g. PLK1, CCNB1/2), since the down-regulation was transient and not accompanied by reduced protein levels. However, the persistent phosphorylation of CHEK1 after 4 Gy suggested a role for CHEK1 in the late arrest, consistent with the abrogation of the arrest in CHEK1-depleted cells. TP53 was not necessary for the late G(2)-arrest, but mediated an intermediate arrest (2-10 hours after IR) independently of ATM and CHEK1. In conclusion, the IR-induced arrest in G(2) is mediated by ATM immediately after irradiation, with TP53 for independent and transient back-up, while CHEK1 is necessary for the late arrest.

Defective expression of genes involved in mitotic chromosome segregation (e.g. AURKA, BUB1B), DNA damage response (e.g. TP53, BRCA1), and telomere function (e.g. TERT) may play a role in the development of tumor aneuploidy.The levels of TP53, BRCA1 and TERT were assessed in 55 sporadic colorectal tumors and 37 normal mucosas using tissue microarrays and immunohistochemical detection, and their associations with DNA aneuploidy, levels of mitotic spindle proteins AURKA, AURKB, MAD2L1 and BUB1B and clinicopathological parameters were investigated.DNA aneuploidy was associated only with TP53 alterations. BRCA1 expression in tumors was significantly correlated with individual mitotic spindle protein expressions, and TERT and MAD2L1 expressions were moderately correlated in the tumor group, suggesting a putative role for TERT in MAD2L1 regulation.Loss of TP53 function appears to be involved in the development of aneuploidy, but not in the deregulation of mitotic spindle protein function.

Background Deproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression. Results We report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression. Conclusion The data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.

The cellular response to chemoradiotherapy was investigated in cells of the HCT116 (wild-type TP53) and HT29 (mutated TP53) human colorectal cancer cell lines to better understand how the chemotherapeutic agent 5-fluorouracil (5-FU) acts as a radiosensitizer in vitro and how it contributes to the well-documented greater efficacy of chemoradiotherapy compared to radiotherapy (or chemotherapy) alone. A bolus 5-FU treatment protocol that simulated actual clinical clearance kinetics was used with a radiation dose given within 90 min after drug addition. The involvements of key signaling pathways (DNA damage response, cell cycle progression, cell proliferation, cell death) in cell responses were investigated concurrently, allowing for direct correlations of numerous treatment response phenotypes. Early DNA damage response, substantial cell death, loss of clonogenicity, and senescence characterized both radiotherapy- and chemoradiotherapy-treated cultures but not chemotherapy-treated cultures. The largest G(2)/M arrests and strongest correlation of senescence with non-clonogenicity were seen in radiotherapy- and chemoradiotherapy-treated HCT116 cell cultures, suggesting that functional TP53 could play a role in maintaining/inducing these cellular phenotypes. Overall, chemoradiotherapy proved to be the most effective treatment modality since it resulted in the strongest growth inhibitions, largest G(2)/M arrests, largest fractions of senescent cells, and complete loss of clonogenicity in both cell lines.

Interleukin (IL)-33 is a novel member of the IL-1 family of cytokines that promotes Th2 responses in lymphocytes as well as the activation of both mast cells and eosinophils via the ST2 receptor. Additionally, IL-33 has been proposed to act as a chromatin-associated transcriptional regulator in both endothelial cells of high endothelial venules and chronically inflamed vessels. Here we show that nuclear IL-33 is expressed in blood vessels of healthy tissues but down-regulated at the earliest onset of angiogenesis during wound healing; in addition, it is almost undetectable in human tumor vessels. Accordingly, IL-33 is induced when cultured endothelial cells reach confluence and stop proliferating but is lost when these cells begin to migrate. However, IL-33 expression was not induced by inhibiting cell cycle progression in subconfluent cultures and was not prevented by antibody-mediated inhibition of VE-cadherin. Conversely, IL-33 knockdown did not induce detectable changes in either expression levels or the cellular distribution of either VE-cadherin or CD31. However, activation of endothelial cell cultures with either tumor necrosis factor-alpha or vascular endothelial growth factor and subcutaneous injection of these cytokines led to a down-regulation of vascular IL-33, a response consistent with both its rapid down-regulation in wound healing and loss in tumor endothelium. In conclusion, we speculate that the proposed transcriptional repressor function of IL-33 may be involved in the control of endothelial cell activation.

Loss of TP53 function may contribute to 5-fluorouracil (5-FU) resistance in colorectal cancer since TP53-deficient cells may be unable to undergo apoptosis in response to 5-FU-induced DNA damage. 5-FU treatment of TP53-deficient cells would provide useful information on the apoptotic response to drug-induced DNA damage in the absence of TP53 and its transcriptional targets. We investigated apoptosis induction and cell cycle alterations in response to short-term treatment with two different 5-FU concentrations following siRNA-mediated knockdown of TP53 in the TP53-proficient HCT116 colon cancer cell line. We focused on high-dose 5-FU treatment to investigate the apoptotic phenotype in 5-FU-treated cultures since this dose resulted in apoptosis induction at 24 h of treatment, whereas clinically-relevant bolus 5-FU treatment of HCT116 cultures did not. Gene expression alterations were also assessed in 5-FU-treated HCT116 cultures using whole genome expression arrays. Compared to 5-FU-treated TP53-proficient HCT116 cultures, 5-FU-treated TP53-depleted HCT116 cultures showed lack of CDKN1A induction, decreased apoptotic levels, decreased FAS and TNFRSF10B transcript levels and cleaved PARP protein levels, G1/S transition arrests, decreased CCND1 protein levels, and smaller intra-S phase arrests. Alterations in gene expression in 5-FU-treated TP53-depleted HCT116 cultures confirmed previously-reported TP53 target genes and suggested potentially novel TP53 target genes (e.g. APOBEC3C, BIRC3, JMJD2B, LAMP3, MYO1E, PRRG1, SULF2, TACSTD2, TncRNA, ZFYVE20) that may play a role in mediating the 5-FU-induced DNA damage response in TP53-proficient cells. Abrogation of TP53 function in 5-FU-treated HCT116 cultures results in reduced apoptosis, TP53- and CDKN1A-independent G1/S phase arrests that may be protective against apoptosis, smaller intra-S phase arrests, and transcript level decreases of both reported TP53 target genes as well as potentially novel TP53 target genes.

The spindle checkpoint, the primary mechanism to ensure that two daughter cells receive the same amount of DNA, is compromised in many malignant tumors and has been implicated as a contributor to aneuploidy and carcinogenesis. The extent of expression and subcellular localization of the spindle proteins Aurora A, Mad2, and BUBR1 varies considerably in different immunohistochemical (IHC) reports from archival tumor tissues. Given the conflicting reports in the literature about the localization of these proteins, we examined the subcellular localization of Aurora kinase A, Mad2, and BUBR1 in normal and cancerous human tissues by IHC. In normal tissues, Aurora A was mainly localized to the nucleus when monoclonal or purified polyclonal antibodies were used, and Mad2 was localized to the nucleus, whereas BUBR1 was localized to the cytoplasm. In malignant tissues, Aurora A showed additional staining in the cytoplasm in the majority of tumors analyzed. Furthermore, BUBR1 was also localized to both the nucleus and cytoplasm in a significant fraction of tumors. Subcellular localization of Mad2 was similar in normal and malignant tissues. Thus, the validity of some earlier IHC studies of Aurora A, Mad2, and BUBR1 should be reconsidered, indicating that high-quality antibodies and a high-alkaline antigen-retrieval technique are required to achieve optimal results. We conclude that the subcellular localizations of these spindle proteins are different, although they have overlapping biological functions, and that Aurora A and BUBR1 undergo a shift in the subcellular localization during malignant transformation.

Endostatin is a proteolytic fragment of collagen XVIII that inhibits endothelial cell migration in vitro and experimental tumor growth in vivo. To determine how endostatin affects the in vivo behavior of endothelial cells, we took advantage of a surrogate model of human angiogenesis, in which human endothelial cells are transferred to immunodeficient mice and develop into complex vessels in the course of 30 days. Systemic delivery of human yeast-derived endostatin (serum levels of 30-35 ng/mL) inhibited the number of human vessels dramatically (95% at day 20), as most endothelial cells remained suspended as single cells. The fraction of cells with a migratory phenotype (F-actin–positive, extending pseudopods) was strongly reduced (from 50% to 13% at day 10), while the number of apoptotic and mitotic cells remained unchanged. Endostatin also hampered the recruitment of α-smooth muscle actin–expressing perivascular cells and thus reduced the number of mature vessels (from 64.3% to 28.6% at day 30). Moreover, transcripts of pericyte-recruiting platelet-derived growth factor-B (PDGFB) were strongly reduced in endothelial cells of endostatin-treated mice. Our results are strong evidence that endostatin inhibits angiogenesis at several levels in vivo, including perivascular cell recruitment.

The colon mitosis inhibiting peptide pyroglutamyl-histidyl-glycine (pEHG) increases the expression of c-fos, fosB and egr-1 genes in the colon carcinoma cell line HT-29. However, the effect on non-tumorigenic colonic cells has not been investigated.After exposure of the cell lines YAMC (from colon mucosa of Immorto mice) and IMCE (fromn Immorto-Min mouse hybrid) to pEHG, DNA-synthesis was analysed by H3-thymidine incorporation, apoptosis and necrosis by fluorescence microscopy, and cell cycle distribution by flow cytometry.pEHG inhibited DNA-synthesis with a maximal effect at 10(-8)-10(-9) M, but stimulated at 10(-4) M. It blocked cell flow through the cell cycle at GC/M after 8 h of treatment, but had no effect on apoptosis or necrosis at any concentration. A low concentration of ascorbic acid stabilised the cells, maybe as a free radical scavanger.pEHG inhibits flux at the G2/M transition, but has no effect on cell death.

Colorectal carcinomas are characterized by frequent recurrent gains and losses of chromosomal material, especially gains of chromosome arms 20q and 13q, and losses of chromosome arms 18q and 4q. These may be important in the development and progression of colorectal carcinomas. Chromosomal aberrations detected by comparative genomic hybridization in 67 sporadic colorectal carcinomas were examined for their possible associations with patient survival. Dukes' stage, tumor DNA ploidy status, and TP53 genotype/phenotype were also examined for the same. Patients with losses of chromosomal arms 1p, 4q, 8p, 14q, or 18q or gain of chromosomal arm 20q had significantly shorter survival times than those without these aberrations (univariate relative risk 3.45, 2.71, 3.32, 3.26, 3.32, 3.91, respectively), as did patients with more than six chromosomal aberrations per tumor than those with fewer than six aberrations (univariate relative risk 3.26, P = 0.013). DNA aneuploidy and Dukes' stage C + D resulted in poor patient survival (univariate relative risk 3.58, 3.39, respectively). Dukes' stage C + D, 1p loss and 8p loss emerged as the only independent prognostic parameters (relative risk 3.22, 2.53, 2.45, respectively) when entered into multivariate survival analysis together with other significant parameters from univariate survival analysis. Loss of chromosome arm 1p, 4q, 8p, 14q, or 18q or gain of chromosome arm 20q thus results in shortened survival times in colorectal cancer patients. 1p loss and 8p loss were shown to be independent predictors of poor prognosis.

Background Treatment of cells with the anti-cancer drug 5-fluorouracil (5-FU) causes DNA damage, which in turn affects cell proliferation and survival. Two stable wild-type TP53 5-FU-resistant cell lines, ContinB and ContinD, generated from the HCT116 colon cancer cell line, demonstrate moderate and strong resistance to 5-FU, respectively, markedly-reduced levels of 5-FU-induced apoptosis, and alterations in expression levels of a number of key cell cycle- and apoptosis-regulatory genes as a result of resistance development. The aim of the present study was to determine potential differential responses to 8 and 24-hour 5-FU treatment in these resistant cell lines. We assessed levels of 5-FU uptake into DNA, cell cycle effects and apoptosis induction throughout treatment and recovery periods for each cell line, and alterations in expression levels of DNA damage response-, cell cycle- and apoptosis-regulatory genes in response to short-term drug exposure. Results 5-FU treatment for 24 hours resulted in S phase arrests, p53 accumulation, up-regulation of p53-target genes on DNA damage response (ATF3, GADD34, GADD45A, PCNA), cell cycle-regulatory (CDKN1A), and apoptosis-regulatory pathways (FAS), and apoptosis induction in the parental and resistant cell lines. Levels of 5-FU incorporation into DNA were similar for the cell lines. The pattern of cell cycle progression during recovery demonstrated consistently that the 5-FU-resistant cell lines had the smallest S phase fractions and the largest G2(/M) fractions. The strongly 5-FU-resistant ContinD cell line had the smallest S phase arrests, the lowest CDKN1A levels, and the lowest levels of 5-FU-induced apoptosis throughout the treatment and recovery periods, and the fastest recovery of exponential growth (10 days) compared to the other two cell lines. The moderately 5-FU-resistant ContinB cell line had comparatively lower apoptotic levels than the parental cells during treatment and recovery periods and a recovery time of 22 days. Mitotic activity ceased in response to drug treatment for all cell lines, consistent with down-regulation of mitosis-regulatory genes. Differential expression in response to 5-FU treatment was demonstrated for genes involved in regulation of nucleotide binding/metabolism (ATAD2, GNL2, GNL3, MATR3), amino acid metabolism (AHCY, GSS, IVD, OAT), cytoskeleton organization (KRT7, KRT8, KRT19, MAST1), transport (MTCH1, NCBP1, SNAPAP, VPS52), and oxygen metabolism (COX5A, COX7C). Conclusion Our gene expression data suggest that altered regulation of nucleotide metabolism, amino acid metabolism, cytoskeleton organization, transport, and oxygen metabolism may underlie the differential resistance to 5-FU seen in these cell lines. The contributory roles to 5-FU resistance of some of the affected genes on these pathways will be assessed in future studies.

Background: Established colorectal cancer cell lines subjected to different 5-fluorouracil (5-FU) treatment protocols are often used as in vitro model systems for investigations of downstream cellular responses to 5-FU and to generate 5-FU-resistant derivatives for the investigation of biological mechanisms involved in drug resistance. We subjected HCT116 colon cancer cells to two different 5-FU treatment protocols in an attempt to generate resistant derivatives: one that simulated the clinical bolus regimens using clinically-achievable 5-FU levels, the other that utilized serial passage in the presence of increasing 5-FU concentrations (continuous exposure). HCT116 Bolus3, ContinB, and ContinD, corresponding to independently-derived cell lines generated either by bolus exposure or continuous exposure, respectively, were characterized for growth- and apoptosis-associated phenotypes, and gene expression using 8.5 K oligonucleotide microarrays. Comparative gene expression analyses were done in order to determine if transcriptional profiles for the respective treatment derivatives were similar or substantially different, and to identify the signaling and regulatory pathways involved in mediating the downstream response to 5-FU exposure and possibly involved in development of resistance. Results: HCT116 ContinB and ContinD cells were respectively 27-fold and >100-fold more resistant to 5-FU and had reduced apoptotic fractions in response to transient 5-FU challenge compared to the parental cell line, whereas HCT116 Bolus3 cells were not resistant to 5-FU after 3 cycles of bolus 5-FU treatment and had the same apoptotic response to transient 5-FU challenge as the parental cell line. However, gene expression levels and expression level changes for all detected genes in Bolus3 cells were similar to those seen in both the ContinB (strongest correlation) and ContinD derivatives, as demonstrated by correlation and cluster analyses. Regulatory pathways having to do with 5-FU metabolism, apoptosis, and DNA repair were among those that were affected by 5-FU treatment. Conclusion: All HCT116 derivative cell lines demonstrated similar transcriptional profiles, despite the facts that they were generated by two different 5-FU exposure protocols and that the bolus exposure derivative had not become resistant to 5-FU. Selection pressures on HCT116 cells as a result of 5-FU challenge are thus similar for both treatment protocols.

Background We previously reported that a pool of low molecular weight peptides can be extracted by alkali treatment of DNA preparations obtained from prokaryotic and eukaryotic cells after intensive deproteinization. This class of peptides, isolated from wheat bud chromatin, induces growth inhibition, DNA damage, G2 checkpoint activation and apoptosis in HeLa cells. In this work we studied their mechanism of action by investigating their ability to interfere with DNA synthesis. Methods BrdUrd comet assays were used to detect DNA replication defects during S phase. DNA synthesis, cell proliferation, cell cycle progression and DNA damage response pathway activation were assessed using 3H-thymidine incorporation, DNA flow cytometry and Western blotting, respectively. Results BrdUrd labelling close to DNA strand discontinuities (comet tails) detects the number of active replicons. This number was significantly higher in treated cells (compared to controls) from entry until mid S phase, but markedly lower in late S phase, indicating the occurrence of defective DNA synthesis. In mid S phase the treated cells showed less 3H-thymidine incorporation with respect to the controls, which supports an early arrest of DNA synthesis. DNA damage response activation was also shown in both p53-defective HeLa cells and p53-proficient U2OS cells by the detection of the phosphorylated form of H2AX after peptide treatment. These events were accompanied in both cell lines by an increase in p21 levels and, in U2OS cells, of phospho-p53 (Ser15) levels. At 24 h of recovery after peptide treatment the cell cycle phase distribution was similar to that seen in controls and CDK1 kinase accumulation was not detected. Conclusion The data reported here show that the antiproliferative effect exhibited by these chromatin peptides results from their ability to induce genomic stress during DNA synthesis. This effect seems to be S-phase specific since surviving cells are able to progress through their normal cell cycle when the peptide fraction is removed from the culture medium. It is likely that the subsequent apoptosis is a consequence of the failed attempt of the tumour cells to repair the DNA damage induced by the peptides.