4''-O-(omega-Quinolylamino-alkylamino)propionyl derivatives of selected macrolides with the activity against the key erythromycin resistant respiratory pathogens.

Four macrolides-6-O-methyl-8a-aza-8a-homoerythromycin, clarithromycin, azithromycin and azithromycin 11,12-cyclic carbonate, have been selected for the construction of a series of new quinolone derivatives. The quinolone moiety is connected to the macrolide scaffold via a diaminoaklyl 4''-O-propionyl ester chain of varying length. At the terminus the linker is attached via one of the nitrogen atoms in the linker at C(6) or C(7) of the quinolone. Many of compounds described, particularly clarithromycin derivative 37, and azithromycin derivatives 48 and 55, exhibited excellent antibacterial activity against a wide range of clinically relevant macrolide-resistant organisms, with profiles superior to that of telithromycin, an enhanced spectrum ketolide.

NF1 gene loss of heterozygosity and expression analysis in sporadic colon cancer.

BACKGROUND AND AIMS: Colon cancer tumorigenesis is a multistep process of mutation accumulation in a number of oncogenes and tumour suppressor genes. NF1 gene protein, neurofibromin, acts as a tumour suppressor by turning the active form of Ras into an inactive form. This molecular switch has an important role in the control of the cell cycle and differentiation, and changes in Ras activity are present in many different cancers. This is the first study to investigate the role of NF1 in sporadic colon cancer. METHODS: We investigated loss of heterozygosity (LOH) at the NF1 locus. Real time reverse transcription-polymerase chain reaction was used to determine NF1 mRNA expression in tumours and corresponding normal tissue. Expression of neurofibromin was analysed by immunohistochemistry. Relative ratio of NF1 mRNA type I and II isoform expression was also examined. RESULTS: LOH of the NF1 gene was detected in 20.7% of heterozygous samples. NF1 mRNA expression was significantly increased in tumour tissue compared with corresponding normal tissue (p = 0.04291). There was a statistically significant increase in NF1 type I isoform expression (p = 0.0005) in tumour tissue compared with corresponding normal colon tissue. NF1 isoform type II was predominantly expressed in normal tissue while the NF1 isoform type I prevailed in tumour samples. The transition from dominant expression of isoform type II in normal mucous tissue 15 cm away from the tumour to dominant expression of isoform type I in tumour tissue itself was detected. Total neurofibromin expression increased as tumours were more advanced but expression of wild-type neurofibromin remained the same. CONCLUSIONS: Our findings suggest that the NF1 gene may play a role in the development and progression of colon cancer and the NF1 gene may be a potential tumour marker and a new potential target for colon cancer therapy.

nm23-H1 expression and loss of heterozygosity in colon adenocarcinoma.

BACKGROUND: The discovery that genetic alterations in oncogenes and tumour suppressor genes accompany tumour formation in many human tumours has encouraged the search for genes that promote or suppress tumour spread and metastasis; nm23 is a promising candidate for a metastasis suppressing gene. AIMS: To evaluate whether expression of nm23-H1 protein or loss of heterozygosity (LOH) of the nm23-H1 gene is associated with colon cancer progression. MATERIALS/METHODS: Paraffin wax embedded tissue sections were analysed immunohistochemically. DNA isolated from normal and tumour tissue was used for LOH analysis using a variable nucleotide tandem repeat (VNTR) marker located in the untranslated 5' region of the nm23-H1 gene. RNA isolated from tumour and normal tissue was used for "real time" RT-PCR. RESULTS: Of 102 adenocarcinomas examined, 58.8% stained weakly for nm23-H1 protein. There was a negative correlation between nm23-H1 positivity and tumour histological grade. In VNTR analysis, 70.2% of patients were informative and 27.4% of tumours had nm23-H1 LOH. There was a positive correlation between nm23-H1 LOH and both tumour histological grade and Dukes's stage. Expression of nm23-H1 mRNA was increased in 22 of 30 colon tumours compared with normal tissue. No significant correlation was found between nm23-H1 mRNA expression and histological grade or Dukes's stage of tumours. CONCLUSIONS: These findings suggest that nm23-H1 protein expression in early stages may have a role in suppressing metastasis in sporadic colon cancer, whereas at a later stage both reduced nm23-H1 protein expression and LOH of the nm23-H1 gene may play role in colon cancer progression and metastasis.

Submerged gel electrophoresis on Spreadex gels--a new method for APC gene mutation detection.

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease. Patients with FAP develop hundreds to thousands of adenomatous polyps in the colon and rectum during their 2nd or 3rd decades, and one or more of them progress to cancer if left without surgical treatment. The gene responsible for FAP was identified in 1991 and termed the APC (adenomatous polyposis coli) gene. Following identification of APC, a number of germ-line mutations responsible for the development of the disease were found. The purpose of this study was to determine the usefulness of a new method, submerged gel electrophoresis, in the detection of the most-frequent mutation of the APC gene [5-base pair (bp) deletion in codon 1309], especially in the presymptomatic diagnosis of FAP. Genomic DNAs were isolated from peripheral blood of patients and their relatives. We used two methods, electrophoresis on polyacrylamide gel and submerged gel electrophoresis, for the identification of APC gene codon 1309 mutation. After only 110 min PCR fragments of 91 bp and 86 bp (5-bp deletion) were completely resolved on a Spreadex EL300 gel. Our results showed that electrophoresis using Spreadex gels provides a simple and rapid non-radioactive method for determination of the most-frequent germ-line mutations in the APC gene.

Loss of heterozygosity of DPC4 tumor suppressor gene in human sporadic colon cancer.

We investigated the prevalence of DPC4 loss of heterozygosity in sporadic colorectal cancer. Thirty-six cases of human sporadic colon carcinoma and corresponding normal tissue samples were examined to evaluate loss of heterozygosity at the DPC4 tumor suppressor locus using variable nucleotide tandem repeat (VNTR) analysis and three polymorphic markers. From 36 analyzed samples 35 (97%) were heterozygous or informative. Loss of heterozygosity at the DPC4 locus was detected in 18 (51%) of informative tumor DNAs. The DPC4 LOH was more frequent in smaller tumors (<5 cm) than in larger ones. There was no correlation between DPC4 LOH and age or sex of patients. There was a negative correlation between DPC4 LOH and histological grade or Dukes' stage of tumors, but without statistic significance. Observed results are in agreement with the view that malignant progression is consequence of many genetic changes. It can be concluded that inactivation of the DPC4 gene plays a role in a multistep process of outgrowth and progression of colon cancer.

Increased activity of nm23-H1 gene in squamous cell carcinoma of the head and neck is associated with advanced disease and poor prognosis.

The present study was undertaken to determine whether the nm23-H1 gene is expressed in squamous cell carcinoma of the head and neck (SCCHN) and whether the level of nm23-H1 protein or mRNA in cells vary as they progress to a more malignant phenotype. Of the 120 SCCHN studied 54 (45%) stained positively for nm23-H1 protein. Protein expression was significantly higher in more advanced stages of disease. Expression of nm23-H1 was significantly higher in cancer tissues than in normal, adjacent tissue, dysplasia, or carcinoma in situ. The nm23-H1 rate increased with progression of synchronous lesions from dysplasia to carcinoma in situ and finally to carcinoma (P<0.05). Northern blot analyses of tissues with various clinicopathological characteristics also revealed differences in nm23-H1 mRNA expression. When levels of nm23-H1 mRNA were compared to tumor stage, intensity of expression was found to be higher in stages 3 and 4 than stages 1 and 2 (P<0.01). Malignant tumors had a higher level of mRNA nm23-H1 expression than normal or premalignant tissues. The nm23-H1 negative patients survived significantly longer than nm23-H1 positive ones (P<0.05). To study the possible relationship between nm23-H1 gene expression and cell growth rate in tumor cells, the mRNA level in each tumor was compared to proliferative activity. The nm23-H1 gene expression levels were directly related to the [3H]thymidine labeling index in tumor cells (R=0.6681). Our results strongly indicate that the nm23-H1 gene is involved in progression of SCCHN. Together with results obtained on lung cancer, our observations suggest that increased expression of nm23-H1 in cancers of the upper aerodigestive tract may have different implications than elsewhere in the body.

Expression of erbB-3 protein in colorectal adenocarcinoma: correlation with poor survival.

BACKGROUND/AIMS: The family of erbB receptors includes four transmembrane glycoproteins with tyrosine kinase activity. These receptors are widely expressed in normal tissues, but they also have been implicated in the development of several human adenocarcinomas. c-erbB-3/HER-3 has been detected to a greater or lesser extent in many tissues from the digestive, urinary, reproductive and respiratory tracts. The overexpression of c-erbB-3/HER-3 protein has also been shown in 53%-88% of colorectal adenocarcinomas. In this study we investigated the expression of the c-erbB-3/ HER-3 gene product in colorectal tumour samples, and compared the results obtained with several clinicopathological parameters, including the survival of patients. METHODS: Paraffin-embedded tissue sections were analysed immunohistochemically, using monoclonal antibody RTJ1 to human erbB-3 protein. Antibody RTJ1 specificity was confirmed by immunoprecipitation followed by Western blotting analysis. Amplification of the erbB-3 oncogene was tested by dot-blot hybridization. RESULTS: Adenocarcinomas of the colon were positive for erbB-3 protein in 78% of samples examined. Dot-blot analysis showed no amplification of the erbB-3 gene in colon adenocarcinomas. Statistical analysis showed that patients with tumours that could not be stained for erbB-3 protein survived significantly longer (P<0.05) than patients with tumours staining positive for the erbB-3 protein. A Cox proportional-hazards model with stepwise variable selection identified age, sex and erbB-3 expression as important prognostic factors. CONCLUSION: These findings demonstrate that erbB-3 protein expression could serve as a prognostic factor in colorectal malignancies.

Monoclonal antibodies against maternal major histocompatibility complex class I molecules induce rapid abortion in mice.

PROBLEM: The role of antibodies against fetal or maternal antigens in maintaining or losing pregnancy is not clear. METHOD OF STUDY: Term-pregnant mice were injected with monoclonal antibodies against only fetal or fetal and maternal major histocompatibility complex class I molecules. The development of pregnancy was then followed. RESULTS: Antibodies against maternal, but not fetal, major histocompatibility complex class I molecules induced abortion in mice. The abortion occurred 6-8 hr after the administration of autoreactive antibodies. The abortion could only be induced after the formation of placenta. Antibodies against tumor necrosis factor-alpha could not prevent or postpone the abortion. Extensive bleeding has been detected in the placenta of aborting mice 3 hr after the administration of the antibodies. CONCLUSIONS: This study indicates that autoreactive antibodies present risk for pregnancy and that the damage leading to abortion induced by such antibodies most likely occurs at the maternal side of placenta.

Loss of heterozygosity of APC and DCC tumor suppressor genes in human sporadic colon cancer.

We examined 36 cases of human sporadic colon carcinoma and corresponding normal tissue samples to evaluate loss of heterozygosity at the APC and DCC tumor suppressor genes loci using restriction fragment length polymorphism polymerase chain reaction and variable nucleotide tandem repeat analysis. Observed informativity was 83% for APC and 75% for DCC. DNA from 6 (20%) of 30 informative tumors exhibited loss of heterozygosity at the APC locus. Loss of heterozygosity at the DCC locus was observed in 7 (26%) of 27 informative tumor DNAs. Our results support the view that malignant progression is a consequence of more than one genetic change and suggest that inactivation of APC and DCC genes plays a role in a multistep process of colon tumor progression.

The expression of p185(HER-2/neu) correlates with the stage of disease and survival in colorectal cancer.

BACKGROUND & AIMS: HER-2/neu oncogene encodes a transmembrane tyrosine kinase receptor that is amplified and/or overexpressed predominantly in adenocarcinomas. This phenomenon has been most intensively studied in breast carcinoma where its amplification and overexpression correlate with the overall course of disease and poor prognosis. This study was designed to investigate HER-2/neu gene expression in benign and malignant colorectal lesions and to evaluate its prognostic importance in colorectal cancer. METHODS: Two hundred twenty-one samples of normal colon, benign lesions, and colorectal adenocarcinomas were studied for expression of HER-2/neu oncoprotein. Immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections of primary tumor and lymph nodes was performed. Immunoprecipitation followed by Western blotting of freshly frozen samples of the same tumors were also performed. RESULTS: Normal colon mucosa, benign lesions, and adenocarcinomas clearly differed in the expression levels and histological distribution of p185(HER-2/neu). Normal mucosa was mostly negative, but significant number of benign lesions and adenocarcinomas overexpressed HER-2/neu protein. Adenocarcinomas were significantly more positive than benign lesions. The results show significant correlation with the epithelial abnormality degree and clinical parameters including Dukes' classification and relapse-free and postoperative survival period. CONCLUSIONS: The p185(HER-2/neu) rate expression could serve as an independent prognostic factor in patients with p185(HER-2/neu)-positive colorectal malignancies.

Ascorbic acid and 6-deoxy-6-chloro-ascorbic acid: potential anticancer drugs.

The role of ascorbic acid (AA) in prevention and suppression of carcinogenesis has been known for a long time. It was also found that AA may inhibit the growth of some tumor cells in vitro and in vivo. We examined the influence of ascorbic acid and 6-chloro-6-deoxy ascorbic acid (6-Cl-AA) on the growth of various human cell lines: lung fibroblasts (Hef), ovarian adenocarcinoma (OVCAR), colon adenocarcinoma (HT-29), laryngeal carcinoma (HEp2) cells, HEp2 cells resistant to vincristine (HEp2VA3), cervical carcinoma (HeLa) cells, HeLa cells resistant to cisplatin (Helacis), breast adenocarcinoma (SK-BR-3) cells, and SK-BR-3 resistant to doxorubicin (SK-BR-3-Dox), as well as mouse fibroblasts L929, mouse melanoma B16 (Mel B16) cells and Chinese hamster fibroblasts (V79). Both drugs arrested the growth of: HeLa, SK-BR-3, SK-BR-3-Dox, L929, and Mel B16 cells, but did not influence the growth of others: Hef, OVCAR, HEp2, HEp2VA3 and V79. 6-Cl-AA suppressed more the proliferation of HeLacis, SK-BR-3-Dox and Mel B16 cells than AA, while AA was active only against HT-29 cells. Inhibitory effect of 6-Cl-AA was confirmed by the in vivo experiments on solid melanoma B16 tumors. Our results indicate that AA and 6-Cl-AA could serve as potential antitumor agents, especially against some tumor cells resistant to chemotherapy.

[Genetic basis of tumors of the colon]. / Geneticka osnova tumora debelog crijeva.

Recent advances in understanding the genetic basis of malignant disease have been dominated by research in colorectal cancer. It has been postulated in previous studies that colorectal adenomas and cancer occur in several rare inherited syndromes and more commonly as sporadic cases. However, new evidence suggests that inherited susceptibility may also be important in a large fraction of so-called sporadic cases. These discoveries will, in very near future, inevitably lead to radical changes in the clinical management of this disease, particularly with the introduction of molecular diagnostic procedures, new chemoprevention protocols, and possibly gene therapy. This review discusses the current developments in colorectal cancer genetics which will be central to such changes.

nm23-H1 gene expression in ovarian tumors--a potential tumor marker.

Ovarian carcinomas that have a distinctive natural history with early dissemination are particularly problematic. The aim of this immunohistochemical study was to assess whether the nm23-H1 gene product, which in some tumors shows inverse association with metastatic potential, could serve as a prognostic marker for ovarian carcinomas. The study, based on 73 benign and 54 malignant ovarian tumors, showed clear differences in the frequency of nm23-H1-positive samples, the intensity of staining and the histological localization of this protein. Differences were observed between normal ovary samples and benign lesions as well as between benign tumors and ovarian carcinomas and were highly significant. Furthermore, carcinomas that had detectable metastasis at the time of surgery were negative for nm23-H1 protein more frequently than those that did not. Although this is a prospective study in which collection of clinical data is ongoing, the results strongly suggest that nm23-H1 may serve as a potentially valuable marker for ovarian tumors.

Prognostic significance of transforming growth factor alpha TGF-alpha) in human lung carcinoma: an immunohistochemical study.

Despite emerging data relating oncogene expression, growth factors and/or their receptors to the etiology of lung cancer, standard clinicopathological evaluation is still used for the diagnostic and prognostic purposes. Recent studies have shown that expression of some oncogenes and growth factors/receptors may be useful as markers in routine diagnostic and prognostic processes. For example, EGF/erb-B family of peptides may play a role in lung carcinogenesis. Similarly, expression of TGF-alpha mRNA and peptide has been shown to occur in various human lung carcinomas in vivo and in vitro. However, results concerning the role of TGF-alpha in lung carcinoma are conflicting and therefore its clinical value still remains obscure. To better evaluate the potential value of TGF-alpha in clinical application we have investigated the relationship between TGF-alpha expression in 51 lung carcinomas and 26 different clinical and clinicopathological parameters. The only significant correlation noted was between TGF-alpha and venous blood erythrocytes and eosinophils. This study suggests a relationship between metastasis and aggressive behavior of lung cancer. This data shows that TGF-alpha expression can not serve as an independent tumor marker for lung cancer.

p185neu is expressed in yolk sac during rat postimplantation development.

We have shown that the neu oncogene product (p185neu) is not present in the rat embryo before organogenesis. However, coincident with the onset of organogenesis, p185neu was detected in neural and connective tissue as well as in the secretory epithelium as was described by Kokai et al. (1987). In addition, p185neu is also expressed in the rat visceral yolk sac (VYS) endodermal cells but not in the mesenchymal and mesothelial layers of the same structure nor in the amnion. The first detectable sign of p185neu expression in VYS was found at d 11 of gestation and the levels of protein increased towards the end of pregnancy. In the yolk sac carcinoma (YSC), which is considered to be the malignant counterpart of the rat yolk sac, p185neu was observed only within columnar epithelial cells (the visceral component of the neoplasm) while parietal endoderm-like cells were devoid of detectable protein. From d 9 of pregnancy up to delivery some of the trophoblastic giant cells also showed a faint to moderate immunoreactivity. Results are presented which would indicate a possible role of p185neu in rat embryogenesis.

Immunohistochemical detection of TGF-alpha, EGF-R, c-erbB-2, c-H-ras, c-myc, estrogen and progesterone in benign and malignant human breast lesions: a concomitant expression.

The expression of transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGF-R) and oncogenes c-erbB-2, c-H-ras, c-myc, as well as estrogen (ER) and progesterone (PR) receptors were studied immunohistochemically in the tissue of 21 benign and 58 malignant human breast lesions. Twenty nine (50%) of 58 carcinomas were positive for EGF-R and c-erbB-2 product, 55 (94.8%) for c-myc product, 9 (15.5%) for c-H-ras product and 17 (29%) for TGF-alpha. Eighteen of 58 (31%) carcinomas were estrogen receptor positive and 22 (38%) were positive for progesterone receptor. No correlation was found between expression of each investigated parameter and the clinical stage or degree of histological differentiation of the carcinomas. However, a significant positive correlation was observed between lymph node involvement and c-erbB-2 and EGF-R/c-erbB-2 positive tumors. A strong correlation was also observed between high levels of EGF-R and low levels of estrogen receptor. In 15 of 17 cases we found simultaneous expression of EGF-R and TGF-alpha. We also found interesting patterns in concomitant expression of the investigated parameters suggesting a possible cascade of events that occur in breast cancer cells.

High c-erbB-2 protein level in colorectal adenocarcinomas correlates with clinical parameters.

Sections of normal colon, adenomas, and adenocarcinomas were examined by immunohistochemistry for the expression of c-erbB-2 proto-oncogene product in order to assess its potential diagnostic value in predicting the malignant potential of these lesions. We compared the degree of epithelial abnormality and clinical parameters, including Dukes' classification and survival time with the extent of immunoperoxidase staining. Sections of normal colon and tubular adenomas examined demonstrated a weak immunostaining localized to the luminal surface cells. However, higher level of c-erbB-2 expression was observed in dysplastic areas of one tubular and one villous adenoma. Out of 40 adenocarcinomas, only 2 samples showed weak immunoreaction, while 38 samples were moderately or strongly positive for c-erbB-2 protein. The intensity of staining correlated positively with the stage of disease and postoperative survival time.