Cloning and characterisation of the RBCC728/TRIM36 zinc-binding protein from the tumor suppressor gene region at chromosome 5q22.3.

DNA alterations at chromosome 5q22 occur frequently in different types of tumors including urological cancers. Previously, we narrowed a common target region between loci D5S659 and D5S2055 at chromosome 5q22.3 by microsatellite allelotyping. After constructing a BAC contig and shot-gun sequencing we identified a putative exon by the NIX software package. By PCR cloning using the putative exon (5qex5) specific primers and primers directed to the vector (lambdaZAPII) sequence of human brain and kidney cDNA libraries, we obtained a full-length cDNA of 4074 bp of the new gene RBCC728/TRIM36 (GenBank accession no.) with an ORF coding for a protein of 728 amino acids. The TRIM36, which is a new member of the tripartite motif (TRIM) gene family, shows a RING finger C3HC4 structure, two B-box, a coiled-coil, a fibronectin type III and a C-terminal domain of unknown function (SPRY). The TRIM36 has a weak homology to MID1. Immunohistochemistry of recombinant and native TRIM36 displays a cytoplasmic, slightly filamentous staining pattern in COS-7 cells. The TRIM36 is expressed in adult testis, brain, prostate, kidney, heart and lung. A variable level of TRIM36 expression was detected by Q-RT-PCR in conventional RCC, while the gene was consequently upregulated in PCs. We did not find mutation in the open reading frame of the TRIM36 in cancer cells. The overexpression of the TRIM36 in the vast majority of prostate cancer suggest that this gene might be involved in the prostate tumorigenesis.

Frequent allelic changes at chromosome 7q34 but lack of mutation of the BRAF in papillary renal cell tumors.

We have analyzed the BRAF locus on chromosome 7q34 with microsatellites for allelic changes and exons 11 and 15 of the BRAF with sequencing for mutations in 50 kidney cancers including 20 papillary, 15 conventional and 15 chromophobe renal cell carcinomas (RCC). Allelic changes at the BRAF locus were seen in 16 of the 20 papillary, 3 of the 15 conventional RCCs and 2 of the 15 chromophobe RCCs. Sequencing failed to disclose mutations in exons 11 and 15 of the BRAF gene in any of the tumors. Our data indicate that BRAF mutation does not play a role in the development of renal cell tumors.

Detection of n-myc gene amplification in neuroblastoma by comparative, in situ,and real-time polymerase chain reaction.

We have used semiquantitative comparative and real-time quantitative polymerase chain reactions (PCR) to detect n-myc gene-amplification in 20 frozen neuroblastoma biopsies and IMR 32 cell line to predict biological behavior of the tumors. Two primer pairs were used for the semiquantitative method to co-amplify a 520-bp fragment of the beta-globin gene--used as a single copy reference standard--and a 258-bp fragment of the n-myc gene. After 30 cycles the PCR products were electrophoresed through an agarose gel and were compared to each other with use of a gel-densitometer. Real-time quantitative analyses were performed in a LightCycler instrument. A single primer pair was used to amplify a 120-bp fragment of the n-myc oncogene and a LC640-labeled fluorescent probe pair to detect the product. Calibration curve, set up from a serial dilution including samples with 1, 2, 10, 13, 25-fold n-myc oncogene amplification, was used for quantitative analysis. The semiquantitative method did not show distinct difference between tumor groups with no amplification and less than 10-fold amplification, whereas quantitative LightCycler analysis was able to detect even 2-fold amplification. Differentiated neuroblastomas seldom show n-myc amplification. In spite of this, we have found two partly differentiated tumor samples that contained n-myc amplification. In these cases in situ PCRs were performed to examine the tumor heterogeneity. We used biotinated ATP labeling and the same primer pair as for the LightCycler analysis. In both cases differentiated cells did not show n-myc gene amplification, whereas considerable amplification was detected in the neuroblasts.

Unusual clinical manifestation of virus-associated hemophagocytic syndrome.

A 22-year-old man presented with bilateral painless cervical lymphadenomegaly, difficulties in nasal breathing and bilateral conductive hearing loss. Rhinoscopy and computer tomography disclosed mucosal polyps in the nasal cavity and a polypoid soft mass almost completely filling the whole nasal cavity and the paranasal sinuses. Thoracic and abdominal computer tomography showed mild hepatosplenomegaly and a solitary round lesion in the right lung. No fever or abnormal laboratory parameters were detected. The biopsy from the nasal polypoid lesions and from the cervical lymph nodes showed extensive proliferation of histologically benign erythrophagocytic histiocytes. The diagnosis of virus (Epstein-Barr virus)-associated hemophagocytic histiocytosis was confirmed by immunohistochemical reactions, by polymerase chain reaction, and by Epstein-Barr-Encodes (Early)-RNA in situ hybridization. This case illustrates an unusual clinical manifestation of virus-associated hemophagocytic histiocytosis presenting as mucosal polyps of the upper respiratory tract.

[Causal association between human papilloma virus infection and head and neck and esophageal squamous cell carcinoma]. / Oki összefüggés a humán papillomavírus-fertôzés és a fej-nyaki régió valamint a nyelôcsô laphámrákjának egyes típusai között.

HPVs commonly cause proliferative lesions of squamous epithelium, and infection with certain HPV types carries a high risk of malignant transformation. We used molecular techniques to detect and type HPV in papillomas and carcinomas in the oral cavity and esophagus. DNA was extracted from 150 fresh or paraffin embedded biopsy specimens, and analyzed for HPV by PCR with 15 sets of consensus primers directed to conserved regions of L1 gene, three sets of HPV16E6 primers (specific for the HPV 16 prototype and L83V variant), and sets of primers specific for the E6 gene of other mucosa type HPVs including HPV 6, 11, 16, 18, 52, 58, 66 and 73. Overall, HPV sequences were detected in 61 of 150 specimens. HPV DNA sequences were detected in 16/32 specimens in the oropharyngeal region, in 13/36 specimens in larynx and 32/82 specimens in esophagus. Papillomas contained only the episomal form of HPV 16. In the esophagus, the most common type was HPV 73. In all specimens examined, HPV 6/11 (4/150), HPV 16 (23/150), HPV 35 (1/150), HPV 45 (1/150), HPV 54 (1/150), HPV 58 (1/150), HPV 61 (1/150), HPV 66 (1/150), HPV 68 (2/150), HPV 70 (3/150), HPV 72 (1/150), HPV 73 (16/150), double HPV infection (2/150), and unidentified HPV type (4/150) was detected. Interestingly, HPV was found in all verrucous carcinomas and in 18/22 basaloid squamous cell carcinomas. HPV16E6 T350G mutant were observed only in two of eight carcinomas. Using correspondence analysis, a segregation of specific virus types in specific clinico-pathologic lesions (verrucous carcinoma and basaloid squamous cell carcinoma) was proved. It was shown that the relative rates of the HPV positive tumors were significantly higher in women than in men. The synergic action of mucosal irritation and HPV infection may be necessary for the development of the papillomas and the specific types of carcinomas in the oral cavity and in the esophagus.

[Detection of n-myc gene amplification in neuroblastoma using polymerase chain reaction based methods]. / N-myc-amplifikáció vizsgálata neuroblastomában PCR-alapú módszerekkel.

We have used semiquantitative and real-time quantitative PCRs to detect n-myc gene-amplification in 21 frozen neuroblastoma biopsies and IMR 32 cell line in order to predict biological behaviour of the tumors. Two primer pairs were used in the semiquantitative method to co-amplify a 520-bp fragment of the beta -globin gene -used as a single copy reference standard -and a 258-bp fragment of the n-myc gene. After 30 cycles the PCR products were electrophoresed through an agarose gel and were compared to each other with use of a gel-densitometer. Real-time quantitative analysis was performed in a LightCycler instrument. A single primer pair was used to amplify a 120-bp fragment of the n-myc oncogene and a LC640-labelled fluorescent probe pair to detect the product. Calibration curve, which was set up from a serial dilution including samples with 1, 2, 10, 13, 25-fold n-myc oncogene amplification, was employed for quantitative analysis. Semiquantitative method did not show distinct difference between tumor groups with no amplification and less than 10-fold amplification, while quantitative LightCycler analysis was able to detect even 2-fold amplification. In situ PCRs were performed in two cases of differentiated tumor samples which contained n-myc amplification. We used biotinylated ATP labelling and the same primer pair as for the LightCycler analysis.In both cases differentiated cell forms did not show n-myc gene amplification, while considerable amplification was detected in the neuroblasts.