George Mariatos

Transcription factor E2F‐1 acts as a growth‐promoting factor and is associated with adverse prognosis in non‐small cell lung carcinomas

Numerous upstream stimulatory and inhibitory signals converge to the pRb/E2F pathway, which governs cell‐cycle progression, but the information concerning alterations of E2F‐1 in primary malignancies is very limited. Several in vitro studies report that E2F‐1 can act either as an oncoprotein or as a tumour suppressor protein. In view of this dichotomy in its functions and its critical role in cell cycle control, this study examined the following four aspects of E2F‐1 in a panel of 87 non‐small cell lung carcinomas (NSCLCs), previously analysed for defects in the pRb‐p53‐MDM2 network: firstly, the status of E2F‐1 at the protein, mRNA and DNA levels; secondly, its relationship with the kinetic parameters and genomic instability of the tumours; thirdly, its association with the status of its transcriptional co‐activator CBP, downstream target PCNA and main cell cycle regulatory and E2F‐1‐interacting molecules pRb, p53 and MDM2; and fourthly, its impact on clinical outcome. The protein levels of E2F‐1 and its co‐activator CBP were significantly higher in the tumour area than in the corresponding normal epithelium (p<0.001). E2F‐1 overexpression was associated with increased E2F‐1 mRNA levels in 82% of the cases examined. The latter finding, along with the low frequency of E2F‐1 gene amplification observed (9%), suggests that the main mechanism of E2F‐1 protein overexpression in NSCLCs is deregulation at the transcriptional level. Mutational analysis revealed only one sample with asomatic mutation at codon 371 (Glu→Asp) and one carrying a polymorphism at codon 393 (Gly→Ser). Carcinomas with increased E2F‐1 positivity demonstrated a significant increase in their growth indexes (r=0.402, p=0.001) and were associated with adverse prognosis (p=0.033 by Cox regression analysis). The main determinant of the positive association with growth was the parallel increase between E2F‐1 staining and proliferation (r=0.746, p<0.001), whereas apoptosis was not influenced by the status of E2F‐1. Moreover, correlation with the status of the pRb–p53–MDM2 network showed that the cases with aberrant pRb expression displayed significantly higher E2F‐1 indexes (p=0.033), while a similar association was noticed in the group of carcinomas with deregulation of the p53–MDM2 feedback loop. In conclusion, the results suggest that E2F‐1 overexpression may contribute to the development of NSCLCs by promoting proliferation and provide evidence that this role is further enhanced in a genetic background with deregulated pRb–p53–MDM2 circuitry. Copyright

p53 activates ICAM-1 (CD54) expression in an NF-κB-independent manner

Intercellular adhesion molecule‐1 (ICAM‐1) is a crucial receptor in the cell–cell interaction, a process central to the reaction to all forms of injury. Its expression is upregulated in response to a variety of inflammatory/immune mediators, including cellular stresses. The NF‐κB signalling pathway is known to be important for activation of ICAM‐1 transcription. Here we demonstrate that ICAM‐1 induction represents a new cellular response to p53 activation and that NF‐κB inhibition does not prevent the effect of p53 on ICAM‐1 expression after DNA damage. Induction of ICAM‐1 is abolished after treatment with the specific p53 inhibitor pifithrin‐α and is abrogated in p53‐deficient cell lines. Furthermore, we map two functional p53‐responsive elements to the introns of the ICAM‐1 gene, and show that they confer inducibility to p53 in a fashion similar to other p53 target genes. These results support an NF‐κB‐independent role for p53 in ICAM‐1 regulation that may link p53 to ICAM‐1 function in various physiological and pathological settings.

Vascular endothelial growth factor (VEGF) is expressed by neoplastic Hodgkin-Reed-Sternberg cells in Hodgkin's disease

Vascular endothelial growth factor (VEGF) is involved in tumour angiogenesis, an important process for the growth and metastatic potential of solid tumours. Numerous studies have demonstrated up‐regulation of VEGF at both mRNA and protein level in various tumours and a correlation with advanced stage and prognosis has been demonstrated in some cases. Limited information exists about its role in lymphoid malignancies and in particular, Hodgkins disease. The present study examined the immunohistochemical expression of VEGF using the monoclonal antibody VG1 in a series of 61 cases of Hodgkins disease, including both classical Hodgkins disease and the nodular lymphocyte predominance variant, and correlated these results with microvessel density, using an anti‐CD31 monoclonal antibody. In 41 cases (70.6%) of classical Hodgkins disease and one of the three cases of nodular lymphocyte predominance Hodgkins disease, the neoplastic Reed–Sternberg and Hodgkin cells expressed VEGF. The staining observed was cytoplasmic, either diffuse or with a focal paranuclear distribution. Macrophages were always positive, while reactive lymphocytes showed occasional positivity. A variable amount of strong extracellular staining was also observed in the tissue stroma and intravascular plasma staining was prominent. There was no statistically significant relationship between VEGF expression and the subtype of Hodgkins disease or microvessel density. In vitro studies using the Reed–Sternberg cell lines L428 and KM‐H2 were also performed in both normoxia and hypoxia and VEGF protein production was assessed by flow cytometry (FACS), immunoassay of cell culture supernatant, and RT‐PCR. Analysis by FACS demonstrated a subset of cells in both cell lines reacting with VG1 and analysis of secreted VEGF (pg/ml per 1×106 cells) in cell culture supernatant confirmed the normoxic production in both cell lines and significant hypoxic induction (p<0.005). Additionally, both cell lines expressed VEGF mRNA, as demonstrated using the RT‐PCR method. In conclusion, neoplastic cells express VEGF in Hodgkins disease, as is the case in solid tumours, and this expression may be induced by hypoxia. The presence of VEGF in reactive macrophages and in the extracellular matrix might facilitate tumour progression. Copyright

Epithelioid hemangioma (angiolymphoid hyperplasia with eosinophilia) in the oral mucosa: A case report and review of the literature

Angiolymphoid hyperplasia with eosinophilia (ALHE) is a benign uncommon entity whose aetiology and pathogenesis is under debate. Clinically, it is characterised by cutaneous papules or nodules. Cases of this entity reported in the oral mucosa are very rare. We describe such a case, discuss the problems of histological differential diagnosis between ALHE and other diseases of the region and review the literature.

Altered expression of the cell cycle regulatory molecules pRb, p53 and MDM2 exert a synergetic effect on tumor growth and chromosomal instability in non-small cell lung carcinomas (NSCLCs).

BackgroundRecent in vitro studies provide evidence that the cell cycle molecules pRb, p53 and MDM2 form a tightly regulated protein network. In this study, we examined the relationship of this protein network in a series of non-small cell lung carcinomas (NSCLCs), with the kinetic parameters, including proliferative activity or proliferation index (PI) and apoptotic index (AI), and ploidy status of the tumors.Material and MethodsA total of 87 NSCLCs were examined using immunohistochemical and molecular methods in order to estimate the status of the pRb-p53-MDM2 network. The kinetic parameters and the ploidy status of the tumors were assessed by in situ assays. The possible associations between alterations of the network, kinetic parameters and ploidy status of the carcinomas were assessed with a series of statistical methods.ResultsAberrant expression of pRb (Ab) and overexpression of p53 (P) and MDM2 (P) proteins were observed in 39%, 57%, and 68% of the carcinomas, respectively. The comprehensive analysis revealed that concurrent alterations in all three cell cycle regulatory molecules were the most frequent pattern, pRb(Ab)/p53(P)/MDM2(P); this “full abnormal” phenotype represented approximately 27% of the cases. This immunoprofile obtained the highest PI/AI value; whereas, the “normal” phenotype was the lowest one (p = 0.004). Furthermore, the pattern pRb(Ab)/p53(P)/MDM2(P) acquired the highest PI (p < 0.001) and lowest AI (p < 0.001) scores. Interestingly, the groups of carcinomas with impaired expression of one or two molecules attained PI/AI ratio values clustered in a narrow range placed in the middle of the scores exhibited by the “normal” and “full abnormal” phenotypes. These tumors had significantly lower AI, but similar PI values, compared with those noticed in the normal pattern. In addition, it was observed that the pRb(Ab)/p53(P)/MDM2(P) phenotype was also significantly associated with aneuploidy (p = 0.002) and a tendency was observed when the expression of two components was altered (p = 0.055).ConclusionsOur findings suggest that simultaneous deregulation of all members of the pRb-p53-MDM2 network confers an additive effect on tumor growth. The apoptotic pathway seems to be more susceptible to its defects than the cell proliferation machinery. The findings of the ploidy analysis, which are in parallel with those regarding the proliferative activity and the apoptotic rate study, further support the concept that these molecules constitute a tightly regulated network participating in cell cycle control and chromosomal stability.

Expression of p16INK4A and alterations of the 9p21‐23 chromosome region in non‐small‐cell lung carcinomas: Relationship with tumor growth parameters and ploidy status

The 9p21‐23 chromosome region harbors a number of known and putative tumor‐suppressor genes (TSGs). The best characterized gene in this area is p16INK4A (CDKN2A). Alterations of its product have been observed in various malignancies, including non‐small‐cell lung carcinomas (NSCLCs). We earlier investigated the mechanisms underlying p16INK4A inactivation. In the present study, we examined, in a series of 87 NSCLCs, its relationship with the kinetic parameters [proliferation index (PI) and apoptotic index (Al)] and the ploidy status of the tumors. In addition, we extended our previous LOH analysis of the 9p21‐23 region by examining flanking areas of p16INK4A. Aberrant p16 expression was observed in 41.4% of the carcinomas. A significant association was found with increased PI (p = 0.037), but not with apoptosis. Aneuploid tumors were more frequently correlated with abnormal p16 staining (p = 0.05). A high frequency of allelic imbalance (Alm) was noticed at the D9S161 (51.3%) and D9S157 (64.5%) loci, which lie approximately 4cM centromeric and 7cM telomeric, respectively, to CDKN2A. Abnormal p16INK4A expression was strongly correlated with Alm at D9S161 (p = 0.004). Allelic losses at D9S157 occurred more frequently in early stages (p = 0.018) and were significantly associated with deletions at D9S161 (p = 0.035). We conclude that, in a sub‐set of NSCLCs, (i) abnormal p16 expression contributes to tumor growth mainly by increasing the proliferative activity in the initial stages of carcinogenesis; (ii) the association with aneuploidy merely reflects the impact of aberrant p16 on proliferative activity; and (iii) other putative TSGs possibly reside within the 9p21‐23 region that possibly co‐operate in certain cases with CDKN2A in the development of NSCLCs. Int. J. Cancer (Pred. Oncol.) 89:133–141, 2000.

Additional characterization of a hexanucleotide polymorphic site in the first intron of human H-ras gene: comparative study of its alterations in non-small cell lung carcinomas and sporadic invasive breast carcinomas

Intron 1 of the human H-ras gene possesses a polymorphism consisting of repetitions of the GGGCCT consensus. Three alleles have been reported at this locus. We confirmed that two, P1 and P2, display four and two repeats, respectively, with their internal sequence structure similar to that previously described. The third, P3, previously assigned as a three-unit repetition allele according to its electrophoretic mobility and with no other information regarding its internal structure, was also found. Sequence analysis of the P3 allele revealed that it consists of three perfect repeats of the GGGCCT consensus. This polymorphism is present only in human c-H-ras gene, although single hexanucleotide repeats are found scattered within intron 1 of this gene in rodents. Analysis of this locus in matched tumor/distant normal samples from: (i) 38 patients with non-small-cell lung carcinoma (NSCLC), and (ii) 35 patients with sporadic invasive breast carcinoma, revealed: (1) 6.6% and 19% loss of heterozygosity (LOH) respectively, and (2) 10.5% and 2.9% hexanucleotide instability (HI) respectively, detected by the presence of shifted in length alleles. Shifted alleles exhibited altered internal sequence structure in comparison to normal ones, suggesting complex mutational events. The same pattern of alterations was also detected in tissues adjacent to lung adenocarcinomas and dysplasias adjacent to squamous cell carcinomas (7.7% LOH, 5.9% HI), implying that abnormalities at this locus may be early events in lung carcinogenesis. The frequency of alterations (LOH vs. HI) was significantly different among NSCLC and breast cancer (P=.005), probably due to the different tumor biology of each system. Finally, altered mRNA expression of H-ras gene was detected in all cases with HI, but this finding was also observed in samples without HI. In view of reports showing that elements in intron 1 of H-ras gene potentially influence its transcriptional regulation, from our results we cannot exclude that the hexanucleotide locus could be an element with possible involvement in expressional regulation of this gene.

Space weather forecasting at the new Athens center: the recent extreme events of January 2005

From the beginning of this year a new data analysis center [Athens Neutron Monitor Data Processing (ANMODAP) Center] is operated in Athens University producing a real-time prediction of space weather phenomena. At this moment there has been a multi-sided use of twenty-three neutron monitors providing real-time data on the Internet. Moreover, interplanetary space parameters data from Geostationary Orbiting Environmental Satellite and Advanced Composition Explorer (ACE) satellite are also collected in this center. The ANMODAP Center in real-time is of high potential interest, as it is expected to give alerts for ground level enhancements (GLEs) of solar cosmic rays (CRs) and geomagnetic storms and therefore to provide crucial information for Space Weather applications. Forecasting of the last GLE and the geomagnetic variations of CRs on January 2005, is presented.

c-mos immunoreactivity is an indicator of good prognosis in lung cancer

Reports concerning the expression of cytoplasmic components of the mitogen‐activating protein kinase (MAPK) pathway in lung cancer are limited. One of the molecules participating in this pathway is the product of the c‐mos proto‐oncogene. In vitro investigations, in somatic cells, have shown that c‐mos expression has opposing effects on cell cycle progression suggesting that it may represent an important determinant of aberrant cell function. In this study we analysed, by immunohistochemical means, its status in a series of lung carcinomas and correlated the findings with clinicopathological parameters and survival of the patients.

Evaluation of shear bond strength of microwaveable acrylic resins in denture repair: A comparative study

Objective. Acrylic denture base fracture is a common mode of failure. Heat-cured, auto-polymerized, visible light-cured, and microwaveable acrylic resins have been used as repair materials. The aim of this study was to evaluate the shear bond strength of two microwaveable resins (Acron MC and Justi) and one auto-polymerizing acrylic resin (ProBase Cold) as denture repair materials as opposed to a heat-cured one using the non-flasking procedure after thermocycling and photoaging. Material and Methods. Ninety cylindrical specimens were made using the Vertex Rapid Simplified heat-cured acrylic resin. Each repair acrylic resin was poured on the specimens surface using a cylindrical rubber mold with an internal diameter of 8.5 mm. Thirty specimens for each repair material were made. The control group consisted of 10 specimens from each group which were stored in water for 24 h at 37°C; another 10 specimens from each group were subjected to a thermocycling procedure (5–55°C for 1,000 cycles), while the remaining 10 specimens were subjected to a photoaging procedure. Shear bond strength was measured on a universal testing machine and mode of bond failure was examined under a stereomicroscope. Two-way ANOVA and the Bonferroni post hoc test were performed to identify statistical differences at α = 0.05. Results. Justis shear bond values were significantly inferior to those of ProBase Cold (p <0.05) and Acron MC (p <0.05). ProBase Cold and Acron MC acrylic resins exhibited similar values (p >0.05) of shear bond strength. Thermocycling and photoaging did not affect the shear bond values of any of the materials under investigation (p >0.05). Conclusions. ProBase Cold and Acron MC exhibited similar shear bond values. Justi repair material exhibited inferior bond strength compared with that of ProBase Cold and Acron MC. Aging procedures did not affect the bonding properties of any of the repair materials.