Paula M. De Angelis

Hypoxia induces adaptive and reversible gross morphological changes in crucian carp gills

SUMMARY We 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.

Nuclear interleukin-33 is generally expressed in resting endothelium but rapidly lost upon angiogenic or proinflammatory activation.

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.

Comparison of gene expression in HCT116 treatment derivatives generated by two different 5-fluorouracil exposure protocols

BackgroundEstablished 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.ResultsHCT116 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.ConclusionAll 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.

The prognostic value of spontaneous apoptosis, Bax, Bcl‐2, and p53 in oral squamous cell carcinoma of the tongue

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.

Effects of acetaminophen and hydroxyurea on spermatogenesis and sperm chromatin structure in laboratory mice

High doses of acetaminophen (400 mg/kg) or hydroxyurea (200 mg/kg) given intraperitoneally daily for 5 d caused reduction in relative testicular weight in mice (B6C3/F1/BOM M). Testicular atrophy of several tubules was seen in the hydroxyurea-treated mice 5 d after the last exposure, whereas acetaminophen did not lead to such changes. Exposure to acetaminophen caused neither a depletion of glutathione in the testis nor a marked increase in covalent binding. In contrast, significant decreases in the incorporation of thymidine into the testis were observed during the first 3 h following a single treatment with acetaminophen (100 to 400 mg/kg) or hydroxyurea (100 to 200 mg/kg). In mice treated with acetaminophen (400 mg/kg) or hydroxyurea (200 mg/kg) daily for 5 d, flow cytometric analysis revealed large reductions in one of the tetraploid populations of testicular cells (mostly early pachytene spermatocytes) on days 5 and 10. Changes in the populations of the various spermatid stages occurred later; thus, both compounds appeared to cause a delay in spermiogenesis. Indications of abnormal chromatin structure were seen in an increased frequency of vas deferens sperm on days 27 and 33 after the last exposure, when measured as increased susceptibility towards DNA denaturation in situ. In conclusion, high doses of acetaminophen or hydroxyurea inhibit DNA synthesis in the testis. The present data indicate that this leads to reduced testicular weight, a reduction in the number of early pachytene spermatocytes, changes in the proportions of the various spermatid stages, and an apparent alteration in sperm chromatin structure.

Subcellular Localization of the Spindle Proteins Aurora A, Mad2, and BUBR1 Assessed by Immunohistochemistry:

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.

Human papillomavirus (HPV)-positive tonsillar carcinomas are frequent and have a favourable prognosis in males in Norway

Conclusions: This study confirms a high prevalence of human papillomavirus (HPV)-positive tonsillar tumours (52%). The survival of the HPV-positive group was significantly better in males. Objectives: We assessed the prevalence of HPV in 137 patients with tonsillar carcinomas, measured the p53- and Ki-67-positive tumour cell fractions and correlated the results with clinical variables. Patients andmethods: Tumour DNA from patients with squamous cell carcinoma of the tonsillar region was amplified by PCR and sequenced for detection of HPV subtypes. Results: HPV was found in 71/137 (52%) of the tumours; HPV-16 was the most frequent subtype (87%). HPV positivity did not correlate with gender, stage, T- and N categories, Ki-67 expression or p53 positivity. The HPV-positive group had a significantly better survival (p < 0.01) compared with the HPV-negative group in males. In a multivariate analysis HPV status gave prognostic information in addition to the earlier established factors, i.e. age, gender and stage (p < 0.05).

Intratumor chromosomal heterogeneity in advanced carcinomas of the uterine cervix

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.

Chromosomal 20q gain in the DNA diploid component of aneuploid colorectal carcinomas

The order of appearance of different genetic aberrations during the shift from diploidy/near‐diploidy to aneuploidy in colorectal cancers is not yet clear. We studied genetic alterations in flow cytometrically‐sorted DNA diploid and corresponding aneuploid epithelial cell populations from each of 20 colorectal tumors using comparative genomic hybridization, FISH, and PCR. Analysis of the 19 cases in which aberrations were found in the flow‐sorted diploid population indicated that large‐scale aneuploidization in colorectal cancer was preceded by amplification of oncogene(s) localized to chromosome 20q13.2 and by KRAS mutations, but not by TP53 deletions or losses of large chromosomal regions such as 4q, 8p and 18q.

Association of p53 accumulation with TP53 mutations, loss of heterozygosity at 17p13, and DNA ploidy status in 273 colorectal carcinomas

The aim of this study was to establish an experimentally based cutoff level for assessing p53 immunoreactivity in colorectal tumors. The accumulation of p53 protein in 273 colorectal tumors was correlated with previously obtained data on TP53 mutation and loss of heterozygosity at two 17p13 loci in the same tumors. The monoclonal antibody PAb 1801 was used for p53 staining, and the results obtained by immunohistochemistry and immunoblotting were similar. Mutation analyses of exons 5–8 were performed using constant denaturant gel electrophoresis followed by sequencing. There were no statistically significant differences for any measured TP53 gene alteration between the group of tumors without p53-positive nuclei (n = 83) and the group with <5% positive nuclei (n = 58). The majority of mutations within these groups were deletions/insertions and nonsense mutations without p53 accumulation. Therefore, we assume that 5% p53-positive nuclei is the relevant cutoff level to assess TP53 damage in colorectal tumors. A prerequisite for this recommendation is optimal conditions for p53 protein detection. The parameters for p53 dysfunction were correlated to DNA aneuploidy measured by flow cytometry. TP53 mutations were significantly associated with DNA aneuploidy (P <0.00001), and a nonrandom distribution of TP53 gene alterations among diploid (DI = 1), hyperdiploid (1.0 < DI < 1.3), and highly aneuploid (DI > 1.3) tumors indicates that DNA hyperdiploid tumors constitute a separate developmental entity different from tumors with gross aneuploidy.