A novel quantitative method of pten expression assessment in tumor tissue.

Phosphatase and Tensin Homolog deleted on chromosome 10 (PTEN) gene is one of the most important tumor suppressor genes which is involved in the regulation of many signaling cascades (AKT/PKB and MAPK). Subtle changes in its activity lead to cancer susceptibility or aggressive tumor behaviour. Despite the diversity of mechanisms leading to PTEN inactivation, it is frequently associated with a decreased or complete loss of protein expression. About 20% decrease in PTEN expression could lead to the development of cancer. There have been no objective, quantitative methods of PTEN expression assessment that allow to measure the subtle variations of the protein concentration in a tissue-contextual manner. A new quantitative algorithm of immunostaining evaluation based on combination of color deconvolution and relative chromogen signal intensity was used in the study. The proposed algorithm was implemented in the popular ImageJ image analysis software and positively verified in cancer cell lines and tissue models as well as in the tissue samples of colorectal cancer (CRC) patients. The proposed quantitative method of PTEN expression assessment creates an alternative to currently available subjective methods and forms the basis for inter-case and inter-tissue comparisons. Using the algorithm it would be possible to identify three groups of patients with advanced colorectal cancer which could significantly differ in the overall survival. The research should be continued.

Optimization of circulating cell-free DNA recovery for KRAS mutation and HPV detection in plasma.

BACKGROUND: The precise analysis of tumour markers in blood such as circulating cell-free DNA (cfDNA) could have a significant impact in facilitating monitoring of patients after initial therapy. Although high levels of total cfDNA in plasma of cancer patients are consistently demonstrated, a low sensitivity of DNA alterations is reported. OBJECTIVE: The major question regards the recovery of tumour-specific cfDNA such as KRAS mutated DNA and cancer-associated type 16 of human papillomavirus (HPV16). METHODS: TaqMan technology was used for detection of KRAS mutation, HPV16 and to quantify cfDNA in blood plasma. RESULTS: Comparison of four different column-based commercial kits shows that the cfDNA purification carried out by the Genomic Mini AX Body Fluids kit and the QIAamp Circulating Nucleic Acid kit gave us the possibility to improve the sensitivity of detection of KRAS mutation and HPV16. The optimized method was used to follow the reduction in cancer-specific cfDNA after therapy. We found that large volume extractions with low volume of DNA eluate enabled trace amounts of tumour-specific cfDNA from cancer patients to be effectively identified. CONCLUSIONS: Data presented in this study facilitate detection of tumour-specific cfDNA and improve standards needed for the implementation of cfDNA technology into routine clinical practice.

Structure of the 5' region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites.

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5' region of the transcription unit of these genes as well as localization of the 'cis' elements conferring their testis-specific expression gave contradictory results [Widlak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191-200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310-321]. In the present paper we solve these controversies and show that the 5' untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5' UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360 bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on 'cis' regulatory elements localized within exon 1 and the intron sequences.

Estimation of risk of inherited medullary thyroid carcinoma in apparent sporadic patients.

PURPOSE: The study was undertaken to evaluate the frequency of inherited medullary thyroid carcinoma (MTC) among patients with apparent sporadic disease. A stepwise algorithm was used depending on clinical indices and the age of patient at MTC diagnosis. PATIENTS AND METHODS: One hundred sixteen patients with MTC verified by postoperative pathologic examination were subjected to genetic analysis of RET exons 10, 11, 13, 14, and 16 by means of polymerase chain reaction, restriction endonuclease digestion, and DNA sequencing. RESULTS: Among 116 apparent sporadic MTC patients, we identified eleven (9.5%) RET germline mutation carriers. Seven of these (6.0%) were found by routine analysis (exons 10 and 11). The frequency of inherited disease among patients younger than 45 years at diagnosis was 10.2% by analysis of typical mutations in exons 10 and 11. Extended genetic analysis (sequencing of exons 11, 13, 14, and 16) yielded 6.1% additional diagnoses, giving a risk of 16.3% in this age group. One previously unreported mutation in exon 11 affected codon 649 (TCG>TTG, Ser>Leu). In the true sporadic MTC patients younger than 30 years at diagnosis, frequencies of 36% and 4.5% in polymorphic variants L769L and S836S, respectively, were observed. The frequency for L769L was higher than in older patients (P <.05). CONCLUSION: The frequency of inherited disease among apparent sporadic medullary thyroid carcinoma patients is close to 10% in the Polish population of MTC patients. The extended analysis of all known RET proto-oncogene mutation sites is obligatory in patients younger than 45 years at diagnosis, but we also see the need to analyze the impact of rarer mutations in older patients.

Identification of a microsatellite region composed of a long homopurine/homopyrimidine tract surrounded by AT-rich sequences upstream of the rat stress-inducible hsp 70.1 gene.

A DNA region containing several repetitive motifs has been detected about 1.9 kbp upstream of the transcription unit of the rat stress-inducible hsp 70.1 gene. The most interesting element of this area is a microsatellite sequence (GA)6CAG(TC)24 that consists of an inverted repeat partially overlapping with the long homopurine/homopyrimidine tract (Pu/Py). DNA molecule within the described sequence can theoretically adopt alternate, non-B structures (H-DNA or cruciform) containing single-stranded regions. This microsatellite region is flanked by AT-rich sequences containing several poly(A) tracts. The longest of them with a possible potential to destabilized a double-stranded DNA helix is localized around 160 bp downstream the (GA)6CAG(TC)24. The DNA fragment containing sequences described above was subcloned into the pUC19 vector and the resulting plasmid was subjected to the standard S1 susceptibility assay. Preliminary mapping of the S1 cleavage site indicates for the formation of the non-B-DNA structure within the Pu/Py tract. This is to our knowledge a first report on the existence of a complex microsatellite region on upstream the 5'-end of the hsp 70 gene in mammals.

Expression of the testis-specific HSP70-related gene (hst70 gene) in somatic non-testicular rat tissues revealed by RT-PCR and transgenic mice analysis.

The hst70 gene which belongs to rat HSP70 multigene family is highly expressed in pachytene spermatocytes. Using a transgenic mice model it was found that the promoter of the rat hst70 gene directs the expression of the chloramphenicol acetyl transferase (CAT) reporter gene not only to testis but also to multiple somatic tissues. In wild-type rats the expression of the hst70 gene in tissues other than testis was confirmed by non-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Beside the testis, the CAT expression in transgenic mice and the hst70 gene transcripts in wild-type rats were found in brain, pituitary, epididymis, vas deferens, adrenals, spleen, lung, ovary, oviduct and uterus. The only tissue in which both the CAT expression and the hst70 gene activity has not been found was the liver. These observations suggest possibly more universal, not confined to spermatogenesis, function of the hst70 gene.

Construction of cDNA library from liver RNA of heat shocked rats and DNA sequence analysis of the clone containing the 3'-end untranslated region (3'UTR) of the heat inducible gene hsp70.2.

cDNA library constructed from liver RNA of rats subjected to hyperthermia was used to isolate divergent 3' end untranslated regions (3'UTR) of heat inducible hsp70.1 and hsp70.2 genes. As a result of a double selection procedure with the use of DNA-DNA hybridization and PCR analysis 9 clones containing cDNA sequences derived from the 3'UTR of the hsp70.2 gene were selected. Nucleotide sequence of the cloned inserts was established and the Northern blot analysis was performed to identify the heat inducible transcript encoded by the hsp70.2 gene.