D.A. Spandidos

Activation of RAS family genes in urothelial carcinoma.

PURPOSEnBladder cancer is the fifth most common malignancy in men in Western society. We determined RAS codon 12 and 13 point mutations and evaluated mRNA expression levels in transitional cell carcinoma cases.nnnMATERIALS AND METHODSnSamples from 30 human bladder cancers and 30 normal tissues were analyzed by polymerase chain reaction/restriction fragment length polymorphism and direct sequencing to determine the occurrence of mutations in codons 12 and 13 of RAS family genes. Moreover, we used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profile of RAS genes in bladder cancer specimens compared to that in adjacent normal tissues.nnnRESULTSnOverall H-RAS mutations in codon 12 were observed in 9 tumor samples (30%). Two of the 9 patients (22%) had invasive bladder cancer and 7 (77%) had noninvasive bladder cancer. One H-RAS mutation (11%) was homozygous and the remaining 89% were heterozygous. All samples were WT for K and N-RAS oncogenes. Moreover, 23 of 30 samples (77%) showed over expression in at least 1 RAS family gene compared to adjacent normal tissue. K and N-RAS had the highest levels of over expression in bladder cancer specimens (50%), whereas 27% of transitional cell carcinomas demonstrated H-RAS over expression relative to paired normal tissues.nnnCONCLUSIONSnOur results underline the importance of H-RAS activation in human bladder cancer by codon 12 mutations. Moreover, they provide evidence that increased expression of all 3 RAS genes is a common event in bladder cancer that is associated with disease development.

Human papillomavirus infection of non-melanoma skin cancers in immunocompetent hosts

Human papilloma viruses (HPVs) consist of more than 70 different types and are known to be associated with numerous malignant tumors, including carcinomas of the mucosal and cutaneous epithelium. Non-melanoma skin carcinoma (NMSC) is the most frequently occurring malignancy worldwide in the Caucasian population. Most studies examining the involvement of papillomaviruses in the development of cutaneous carcinomas have been performed on lesions from patients with epidermodysplasia verruciformis or from immunosuppressed patients. Our specimens were obtained from 108 immunocompetent patients with benign and malignant skin lesions, and HPVs were detected in 27%. HPV 8 and HPV 18 were the most frequent types (62 and 48%, respectively). Our results suggest that HPVs, particularly the oncogenic potential of certain types such as HPV 8, 18, and 5 could contribute to the development of NMSCs.

Genomic instability, mutations and expression analysis of the tumour suppressor genes p14ARF, p15INK4b, p16INK4a and p53 in actinic keratosis

Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). We investigated the involvement of the CDKN2A, CDKN2B and p53 genes in AK and in the progression of AK to SCC. Mutational analysis on exons 1a, 1b and 2 of the CDKN2A locus and exon 1 of the CDKN2B locus as well as allelic imbalance was performed in 26 AK specimens. Expression levels of the genes p14(ARF), p15(INK4b), p16(INK4a) and p53 were examined in 16 AKs and 12 SCCs by real-time RT-PCR. A previously described polymorphism of p16(INK4a) (Ala148Thr) was detected at an allelic frequency of 12%. Six samples carried novel mutations at codon 71 of the CDKN2A locus and one sample presented an additional mutation at codon 65. Two AK samples carried a not-previously described non-UV type missense mutation at codon 184 (Val184Glu) of exon 1b in the p14(ARF) gene. Regarding the CDKN2B locus a new mutation at codon 50 (Ala50Thr) and another at codon 24 (Arg24Arg), were detected. Microsatellite instability (MSI) was found in 15% of AKs in at least one marker, indicating that genetic instability has some implication in the development of AK. Down-regulation of p16(INK4a) and p53 mRNA levels was noted in SCC compared to AK. TSGs expression levels in sun-exposed morphologically normal-appearing skin, suggests that abnormal growth stimuli might exist in these tissues as well. Furthermore, we suggest a possible role of p15(INK4b), independently from the intracellular pathway mediated by p16(INK4a), and of p14(ARF) in AK development, as well as in the progression of AK to SCC. The deregulation of the expression profiles of the CDKN2A, CDKN2B and p53 genes may, independently of mutations and LOH at 9p21, play a significant role in AK and progression of AK to SCC.

Viral DNA detection and RAS mutations in actinic keratosis and nonmelanoma skin cancers

Backgroundu2002 Actinic keratosis (AK) is a well‐established precancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). Basal cell carcinoma (BCC) is a locally aggressive slowly growing tumour that rarely metastasizes. A number of viruses have been proposed to play a role in the development of nonmelanoma skin cancers (NMSC), but the most plausible evidence to date suggests that cutaneous human papillomavirus (HPV) is the key instigating factor.

Hypomethylation along with increased H19 expression in placentas from pregnancies complicated with fetal growth restriction

The expression of imprinted genes is regulated by epigenetic modifications, such as DNA methylation. Many imprinted genes are expressed in the placenta and affect nutrient transfer capacity of the placental exchange barrier. The H19 gene is abundantly expressed by the human placenta and is implicated in the pathogenesis of congenital growth disorders such as Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes. The aim of this study was to investigate the role of DNA methylation on H19 transcription and imprinting, in the pathophysiology of fetal growth restriction (FGR). Thirty one and 17 placentas from FGR-complicated and normal pregnancies were collected, respectively. We studied gene transcription, genotyping and methylation analysis of the AluI H19 on exon 5 polymorphism. Placental expression levels of H19 were significantly increased in the FGR group. The H19 mRNA levels were similar between normal placental samples that demonstrated loss and maintenance of imprinting. Placentas from growth-restricted pregnancies had lower methylation levels compared to normals, in the H19 promoter region. We have demonstrated an increased H19 transcription in the FGR group of placentas. The hypomethylation of the H19 promoters is compatible with the aberrant expression. The association of these two findings is reported for the first time in placental tissues, however, its significance remains unknown. Whether the results of this study represent an adaptation of the placenta to hypoperfusion, or they are part of FGR pathophysiology has to be further investigated.

Detection of Epstein-Barr virus and human papillomavirus in nasopharyngeal carcinoma by the polymerase chain reaction technique

We used the PCR technique to detect the Epstein-Barr virus (EBV) and human papillomavirus (HPV) DNA in paraffin-embedded tissues from Greek patients with nasopharyngeal carcinoma (NPC). The oligonucleotide primers used for the detection of EBV amplify a 375-bp long sequence from the EcoRI B fragment of the viral genome, whereas for HPV the primers amplify a 151-bp long sequence of the viral genome. The PCR products were analysed by agarose gel electrophoresis and visualised by UV illumination after staining with ethidium bromide. Sixty-three specimens were examined. EBV specific sequence was amplified in 20 (32%) and HPV in 12 (19%) out of the 63 samples. There was no co-infection with EBV and HPV. Although there is a high correlation of EBV infection with poorly differentiated NPC in patients from Southern China and South-East Asia, the restricted distribution suggests genetic or environmental cofactors in the development of the neoplasm. Our results confirm this suggestion since there was only a 32% correlation of EBV with NPC in Greece. HPV may also be involved in the carcinogenesis of EBV-negative squamous cell nasopharyngeal carcinomas.

Genetic Instability in Renal Cell Carcinoma

Objective: To investigate the incidence of loss of heterozygosity (LOH) and microsatellite instability (MI) in human renal cell carcinoma (RCC), and to determine a possible activation of H-ras oncogene in these tumours via implication of its polymorphic regions within the first intron and 3′ ends. Methods: In the present study, we investigated the incidence of MI and LOH in 22 RCCs, using a bank of 8 microsatellite markers located on chromosomes 2 (IL1A), 3 (D3S1234), 8 (MYC), 14 (D14S51) and 17 (THRA1, D17S250, D17S579). We also studied the microsatellite DNA of the H-ras oncogene within the first intron (HRM) and the minisatellite DNA of the variable tandem repeat (VTR), which is located 1,000 bp downstream of the H-ras gene and possesses enhancer activity, for genetic instability. Alterations of the 28-bp repetition core were studied employing restriction fragment length polymorphism analysis. Results: MI and LOH were observed in 8 (4 MI and 4 LOH) out of 22 (18%) specimens at 3p21.1-p14.2 and 17q21, indicating the presence of putative tumour suppressor genes (TSGs) at these loci. Alterations of the 28-bp repetition core of H-ras VTR were found in 2 out of 22 cases (9%), while point mutations of the same repetition core were detected in only 1 case (5%). Additionally, 1 case (5%), showed LOH. Conclusions: Our results indicate that genetic instability is a detectable phenomenon in human RCC and it might be associated with the development of the disease. LOH at 3p21.1-p14.2 and 17q21 suggests that important TSGs may be located on these chromosomal regions involved in the tumorigenesis or progression of RCC. Considering the fact that the DNA sequence of this VTR region contains a target area for transcription and other regulation factors of H-ras gene expression, these findings could be of importance as regards the involvement of this gene in the process of carcinogenesis in RCC.

Deregulation of the tumour suppressor genes p14ARF, p15INK4b, p16INK4a and p53 in basal cell carcinoma

Backgroundu2002 Basal cell carcinoma (BCC) is a locally aggressive slowly growing tumour that rarely metastasizes and is mostly seen in older members of the population.

No correlation between ras, c-myc and c-jun proto-oncogene expression and prognosis in advanced carcinoma of cervix.

55 patients suffering from stage III or IV carcinoma of cervix were treated with two pulses of neo-adjuvant chemotherapy prior to radical radiotherapy. 51% (26/51) had a partial response. The initial response to chemotherapy is associated with significantly better long-term survival. The 3-year survival of chemotherapy responders is 62% against 21% for non-responders (P = 0.009 log-rank test). To detect possible differences in oncogene expression in biopsy specimens taken from responding and non-responding patients, paraffin-fixed material was immunocytochemically stained for the expression of the protein products of ras, c-myc and c-jun proto-oncogenes. The frequency of oncogene expression was ras 80.4%, c-myc 45.1% and c-jun 39.2%. There was no statistically significant association between oncogene expression, time to local recurrence or development of metastases or survival.

Role of the angiopoietin/Tie system in pregnancy (Review)

Angiopoietin-1 and -2 are endogenous ligands for the vascular endothelium-specific receptor tyrosine kinase Tie-2. The angiopoietin/Tie system plays a critical role in the regulation of endothelial cell survival and vascular maturation and stability. Apart from its well-established role in vascular morphogenesis, emerging data support the involvement of angiopoietins in inflammation and various malignancies. Previous studies have underlined the significance of several angiogenic factors in normal placental development. In addition, angiogenic imbalance is observed in pregnancy complications related to impaired placentation, such as preeclampsia (PE) and intrauterine growth restriction (IUGR). However, there is only limited information available on the role of the angiopoietin/Tie system in the establishment of a competent feto-maternal vascular system. In this review, we present the current knowledge regarding the role of angiopoietins in normal pregnancy and pregnancy complications.