An epidermal growth factor receptor intron 1 polymorphism in healthy women in Poland.

The frequency of CA allele combinations was assessed in healthy women from Poland and compared to previously published polymorphism data of individuals from Germany and a Caucasian reference group. There were close similarities between these three geographically and ethnically similar populations. By contrast, the distribution of these alleles in European and Asian (Japan) populations proved to be different. There might therefore be major ethnic differences in allelic frequencies of EGFR intron 1 polymorphism. Our results provide new data on EGFR microsatellite instability and may contribute to the understanding of EGFR gene expression regulation. The clinical relevance of these findings warrants further evaluation.

An epidermal growth factor receptor intron 1 polymorphism in healthy women in Poland.

The frequency of CA allele combinations was assessed in healthy women from Poland and compared to previously published polymorphism data of individuals from Germany and a Caucasian reference group. There were close similarities between these three geographically and ethnically similar populations. By contrast, the distribution of these alleles in European and Asian (Japan) populations proved to be different. There might therefore be major ethnic differences in allelic frequencies of EGFR intron 1 polymorphism. Our results provide new data on EGFR microsatellite instability and may contribute to the understanding of EGFR gene expression regulation. The clinical relevance of these findings warrants further evaluation. (Int J Biol Markers 2005; 20: 184-8).

p53 Gene status in relation to ex vivo chemosensitivity of non-small cell lung cancer.

PURPOSE: About 40% of non-small cell lung cancer (NSCLC) patients are candidates for systemic chemotherapy, despite the fact that at diagnosis most NSCLC are usually chemoresistant both in vivo and ex vivo. It is important to develop sufficient methods of prediction of the response to chemotherapy and to find molecular markers that may prognose this response. Therefore, a study on the relationship of p53gene status to the ex vivo chemosensitivity of primary human NSCLC was performed. METHODS: Three drug combinations (carboplatin/etoposide, cyclophosphamide/etoposide/epirubicin, and paclitaxel/carboplatin) were tested in a modified ATP cell viability assay. A group of 28 cases of primary human NSCLC was assessed. RESULTS: Ex vivo chemosensitivity testing showed that tumors with p53 mutations were significantly more resistant to the cyclophosphamide/etoposide/epirubicin regimen than with normal p53 gene ( P = 0.012). However, no correlation was observed for two other treatment regimens. CONCLUSION: Mutations in the p53gene can lead to enhanced chemoresistance, confirming the hypothesis that the p53 gene may serve as a marker of tumor response to treatment in NSCLC. However, the data also illustrate that some additional factors might contribute to drug resistance of the examined tumors.

The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis.

Formalin-fixed, paraffin-embedded (FFPE) tissues are one of the popular sources of diagnostic materials, the easiest to store and transport. They are often used as the source of nucleic acids for retrospective molecular analyses based on DNA amplification by polymerase chain reaction (PCR). However, it is known that nucleic acids from paraffin-embedded tissues are much worse templates than those recovered from fresh tissues. It is exceptionally important in a quantitative analysis, including double differential PCR (ddPCR). Therefore, a pilot study comparing quantity and quality of DNA extracted with various paraffin removal and DNA isolation procedures from FFPE tissues was conducted. Furthermore, the suitability of DNA isolated with optimized procedure for the assessment of erbB-2 average gene copy number (AGCN) was checked. Specimens for comparison of extraction and isolation procedures were generated from the same human normal thyroid tissue embedded in paraffin to eliminate variabilities in tissue processing and sample size. Three procedures of paraffin removal and three procedures of DNA extraction from deparaffinized tissue were compared. Only one procedure provided DNA, which was efficiently amplified in ddPCR. The material obtained with this optimized procedure was used to check the precision of ddPCR by evaluation of AGCN of erbB-2 oncogene. Low variability of obtained results close to expected AGCN value (AGCN=1) indicates high reproducibility of the method, as well as its high accuracy, if the normal value of erbB-2 AGCN in the examined tissue is assumed.

Restricted rotation in chiral peptide nucleic acid (PNA) monomers--influence of substituents studied by means of 1H NMR.

The influence of amino acid side chains [derived from: Ala, Val, Leu, Ile, Phe, Tyr(Bzl), Ser(Bzl), Thr(Bzl), Pro, Trp], incorporated into "aminoalkyl" part of PNA monomers, on the temperature-dependent distributions of rotamers about the tertiary amide bond was studied by means of 1H NMR at 0, 25 and 40 degrees C in CDCl3. The delta G0 values of the energy differences between individual rotamers were calculated. The results may be helpful in the designing of monomers with desirable properties.

Influence of hepatitis C virus (HCV) infection on porphyrin and iron metabolism in porphyria cutanea tarda (PCT) patients.

BACKGROUND: Porphyria Cutanea Tarda (PCT) is the most common form of porphyria. It is characterised by lowered activity of uroporphyrinogen decarboxylase. It seems possible that the hepatitis C virus (HCV) infection triggers the symptoms of PCT. MATERIAL AND METHODS: A group of 29 PCT patients (33-73 years old, mean 54 years) was evaluated. In these patients the serum HCV-RNA (by mean of automatic Cobas AmplicorTM Hepatitis C ver. 2.0 assay, Roche Diagnostics), activity of transaminases (ALT, AST), serum protein fractions, iron, total iron binding capacity (TIBC), ferritin, transferrin, transferrin saturation, and urinary porphyrins excretion were assessed. RESULTS: The presence of HCV-RNA was detected in 12 cases (41.4%). Statistically important differences between currently HCV infected patients and other PCT patients concerned the following parameters: mean body mass (68.5 vs. 78.0 kg, p = 0.02), mean body mass index (22.4 vs. 26.8 kg/m2, p = 0.007), mean serum activities of ALT (112.6 vs. 65.4 U/l, p = 0.02) and AST (85.0 vs. 56.5 U/l, p = 0.038), total serum protein concentration (78.0 vs. 71.6 g/l, p = 0.016) and serum alpha-2 protein concentration (7.2 vs. 5.5 g/l, p = 0.009), respectively. Some other not significant differences were also found: mean serum iron level 182 vs. 169 micrograms/dl, transferrin concentration 2.77 vs. 2.56 g/l, transferrin saturation 43 vs. 45%, serum TIBC 445 vs. 397 mg/dl and ferritin concentration 394 vs. 400 micrograms/l, respectively. The mean urinary uroporphyrin concentrations were 643 micrograms/l vs. 366 micrograms/l (p = 0.16) and those of coproporphyrins were 190 micrograms/l vs. 136 micrograms/l (p = 0.37), respectively. CONCLUSION: In the group of PCT patients investigated no significant relationship between current HCV infection and iron or porphyrin metabolism was noted. Nevertheless, HCV infection significantly influences the symptoms of liver damage and seems to influence other metabolic parameters in PCT patients.

HCV-RNA detection in liver bioptates--a comparison of automatic and 'home-made' protocols combined with a new procedure of HCV-RNA extraction.

BACKGROUND: For HCV-RNA detection in liver tissue a generally accepted reference method has not been established yet. Therefore, we have developed a procedure of HCV-RNA extraction from liver tissue and compared two methods of HCV-RNA detection. MATERIALS AND METHODS: A set of 32 liver biopsy specimens, obtained from chronically HCV infected patients, has been examined. At the time of biopsy, serum HCV-RNA was detectable in 20 patients in RT-PCR automatic Cobas AmplicorTM Hepatitis C assay, ver. 2.0 (Roche Molecular Systems, Inc, Pleasanton, CA, USA) 2 serum samples were negative for HCV-RNA and 10 patients has not been tested. Liver tissue has been homogenized in the presence of CRSR-Green (Fast RNA Kit-Green, Bio101, Inc, Vista, CA, USA) and a mixture of phenol/chloroform/isoamylic alcohol, using a FastPrep homogenizator (Bio101, Inc, Vista, CA, USA). Then RNA has been isolated from the material obtained using the Total RNA Prep Plus procedure (A&A Biotechnology, Gdansk, Poland). Presence of HCV-RNA was next tested by means either of 'home-made' nested RT-PCR procedure or the RT-PCR automatic Cobas AmplicorTM Hepatitis C assay, ver. 2.0. In case of an inhibition of PCR detected in the first run of automatic assay, both PCR procedures have been repeated. RESULTS: In 5 cases of automatic assay an inhibition of PCR reaction has been detected in the first run, the RT-PCR procedures has been then successfully repeated in the second run. The presence of HCV-RNA in the liver tissues was detected in a total of 22 cases (69%) by mean of automatic assay and in a total of 20 cases (63%) by means of 'home-made' RT-PCR procedure. Except for two cases of HCV-RNA positivity in a biopsy tissue, detected by means of automatic but not 'home-made' procedure, the results obtained employing the methods have been identical. In one serum HCV-RNA positive case, the presence of HCV RNA has not been detected in a liver tissue using both methods. CONCLUSIONS: Our procedure of RNA isolation combined either with automatic Cobas AmplicorTM Hepatitis C assay, ver. 2.0 or 'home-made' RT-PCR procedure may be useful for establishing presence of HCV-RNA in liver tissue. In this combination, the automatic assay seems to be more sensitive than the 'home-made' procedure.

Effect of high fiber vegetable-fruit diet on the activity of liver damage and serum iron level in porphyria cutanea tarda (PCT).

BACKGROUND: During the treatment of coronary heart disease with a vegetable-fruit diet, we have observed the positive effect of the treatment on PCT patients. Therefore, we have now examined the short-term results of the diet on the selected PCT activity parameters. The study was approved by our Review Board. MATERIAL AND METHODS: A group of 13 male PCT patients (mean age 52 years) was evaluated. We assessed the body mass index (BMI), serum iron level, activity of transaminases (ALT, AST), severity of skin symptoms, and urinary prophyrins excretions, before and after a three-week period of vegetable-fruit diet. The diet was of natural vegetable/fruit products, and its daily caloric content was ca. 500 kcal/day. RESULTS: The mean BMI before and after the diet period were 26.8 +/- 4.7 vs. 25.8 +/- 4.3 (p = 0.001), the serum activities of ALT 122.0 +/- 60.7 U/l vs. 75.6 +/- 31.8 U/l, and of AST 91.8 +/- 56.0 U/l vs. 55.2 +/- 14.2 U/l (p = 0.001), respectively. The mean serum iron levels were 188.6 +/- 75.7 mg/dl vs. 140.2 +/- 56.4 mg/dl, serum ferritin concentrations 574 +/- 351 vs. 499 +/- 340 ng/ml (p = 0.04), respectively. Severity of skin lesions and urinary coproporphyrins excretion were significantly diminished during the diet; urinary uroporphyrins excretion was also lowered, but not to a statistically significant level. CONCLUSION: In our group of PCT patients, we noticed the beneficial effect of the vegetable-fruit diet on selected disease parameters. The diet may be useful in the treatment of PCT and diseases associated with PCT.

c-myc oncogene gene dosage, serum CEA and CA-15.3 antigen levels, and cellular DNA values in relation to ex vivo chemosensitivity of primary human breast cancer.

A pilot study on relationships of selected molecular factors (c-myc oncogene average gene copy numbers (AGCN); serum CEA and CA 15.3 antigen levels; tumor cells' DNA values), to the ex vivo chemosensitivity of primary female human breast cancer in a modified adenosine triphosphate cell viability chemosensitivity assay (ATP-CVA), was performed. Four drug combinations were tested. A group of 75 cases of female primary breast cancer was assessed. Numerous correlations were found among molecular factors tested but none, with the exception of tumor grading, of these reflected ex vivo chemosensitivity of tumors tested. The results suggest that the parameters tested may not be important factors related to adjuvant chemoresponsiveness of primary human breast cancer to tested drug combinations.

Relationship of c-myc and erbB oncogene family gene aberrations and other selected factors to ex vivo chemosensitivity of ovarian cancer in the modified ATP-chemosensitivity assay.

A pilot study on relationships of selected molecular factors [erbB-1, erbB-2, erbB-3, and c-myc oncogene average gene copy numbers (AGCN); steroid receptors and pS2 gene expression; tumor cells' DNA values] to the ex vivo chemosensitivity of ovarian cancer in a modified adenosine triphosphate cell viability chemosensitivity assay (ATP-CVA), was performed. Despite the relatively small number of patients, numerous correlations among the factors tested were found. Nevertheless, only c-myc gene dosage positively affected ex vivo chemosensitivity of tumors tested.

Lack of squamous cell lung carcinoma in vitro chemosensitivity to various drug regimens in the adenosine triphosphate cell viability chemosensitivity assay.

A pilot study on squamous cell lung carcinoma (LC) chemosensitivity in adenosine triphosphate cell viability chemosensitivity assay (ATP-CVA) was performed. Besides the histological investigation, a modified ATP-CVA was used for the analysis of cancer cell chemosensitivity to four drug regimens, including topotecan, a promising agent for non-small-cell lung cancer (NSCLC) chemotherapy. Results of in vitro chemosensitivity testing showed chemoresistance or only weak response in the predominant amount of tumors.

Amplification of erbB-4 oncogene occurs less frequently than that of erbB-2 in primary human breast cancer.

ErbB-4 protein is a recently discovered member of the ErbB family. The role of ErbB-4 protein in mammary-gland tissue has not been definitively established. To date, the expression of erbB-4 in breast tissue has been determined in only a few cases and, to the best of our knowledge, its amplification has not been examined. We therefore used the double differential polymerase chain reaction (ddPCR) for determination of the amplification profile of erbB-4 and erbB-2, another gene from the ErbB family, in human primary breast cancer specimens. We examined the amplification of the genes in 20 normal breasts and 176 invasive breast cancer samples. Amplification of erbB-2 was detected in 19% and erbB-4 in 13% of the samples studied. Co-amplification of the two oncogenes was found in only five out of 176 samples. Human breast cancer-derived cell lines in most cases overexpress both erbB-2 and erbB-4 (Beerli et al., 1995. Mol. Cell Biol. 15, 6496-6505; Han et al., 1995. Proc. Natl. Acad. Sci. USA 92, 9747-9751), but data on separate erbB-2 overexpression, without overexpression of erbB-4, were also reported (Wosikowski et al., 1997. Clin. Cancer Res. 3, 2405-2414). At the gene level, we found that co-amplification of the genes in the case of human breast cancer is rare. Moreover, an inverse association of the erbB-4 amplification with estrogen receptor activity and direct correlation with the tumor size were found. Due to these correlations, erbB-4 oncogene amplification can be assumed to be of prognostic or predictive value in the diagnosis of breast cancer.

Applications of the Mitsunobu reaction in peptide chemistry.

The Mitsunobu reaction--the nucleophilic substitution of an alcoholic hydroxyl group mediated by the redox system trialkylphosphine/dialkyl azodicarobxylate--is widely used in the chemistry of biologically active compounds. The paper deals with applications of the Mitsunobu reaction in amino acid and peptide chemistry. The process provides easy access to many unnatural amino acids and derivatives. Since the reaction occurs with complete inversion of the configuration at the carbinol chiral centre, it can be used for the synthesis of diastereoisomers of hydroxy- and tioprolines. Cyclization of beta-hydroxy amino acid containing peptides under Mitsunobu reaction conditions leads to a constrained peptide that mimics the stabilizing reverse turn secondary structure.

[Porphyria cutanea tarda--new views on its pathogenesis and therapy]. / Porfiria skórna pózna--nowe spojrzenie na patogeneze i terapie.

Literature data and the results of our studies on the prevalence of the HCV infection markers in patients with porphyria cutanea tarda (PCT) are presented. We also present new views on the PCT pathogenesis in some cases, therapy of the cutaneous changes, and the use of interferon in the patients as a prevention of the hepatocellular carcinoma.