Suppression of tumorigenicity in breast cancer cells by the microfilament protein profilin 1.

Differential display screening was used to reveal differential gene expression between the tumorigenic breast cancer cell line CAL51 and nontumorigenic microcell hybrids obtained after transfer of human chromosome 17 into CAL51. The human profilin 1 (PFN1) gene was found overexpressed in the microcell hybrid clones compared with the parental line, which displayed a low profilin 1 level. A comparison between several different tumorigenic breast cancer cell lines with nontumorigenic lines showed consistently lower profilin 1 levels in the tumor cells. Transfection of PFN1 cDNA into CAL51 cells raised the profilin 1 level, had a prominent effect on cell growth, cytoskeletal organization and spreading, and suppressed tumorigenicity of the stable, PFN1-overexpressing cell clones in nude mice. Immunohistochemical analysis revealed intermediate and low levels of profilin 1 in different human breast cancers. These results suggest profilin 1 as a suppressor of the tumorigenic phenotype of breast cancer cells.

High-Throughput Omics Technologies: Potential Tools for the Investigation of Influences of EMF on Biological Systems.

The mode of action of a huge amount of agents on biological systems is still unknown. One example where more questions than answers exist is covered by the term electromagnetic fields (EMF). Use of wireless communication, e.g. mobile phones, has been escalated in the last few years. Due to this fact, a lot of discussions dealt with health consequences of EMF emitted by these devices and led to an increased investigation of their effects to biological systems, mainly by using traditional methods. Omics technologies have the advantage to contain methods for investigations on DNA-, RNA- and protein level as well as changes in the metabolism.This literature survey is an overview of the available scientific publications regarding biological and health effects of EMF and the application of new high-throughput technologies. The aim of the study was to analyse the amount and the distribution of these technologies and to evaluate their relevance to the risk analysis of EMF. At present, only transcriptomics is able to analyse almost all of the specific molecules. In comparison to ionising radiation, fewer articles dealt with health effects of EMF. Interestingly, most of the EMF articles came from European institutions.Although omics techniques allow exact and simultaneous examinations of thousands of genes, proteins and metabolites in high-throughput technologies, it will be an absolute prerequisite to use standardised protocols and to independently validate the results for comparability and eventually for sound standing statements concerning possible effects of agents like EMF on biological systems.

A defined chromosome 6q fragment (at D6S310) harbors a putative tumor suppressor gene for breast cancer.

Recent evidence obtained by cytogenetic and molecular studies indicates that in breast cancer chromosome 6q is often affected by genetic changes suggesting the existence of putative tumor suppressor genes (TSGs). However the function of gene(s) on this chromosome in breast cancer suppression is not understood. To substantiate further the presence of breast cancer related TSGs at 6q and to define their location, we first performed microcell-mediated transfer of chromosome 6 to CAL51 breast cancer cells for studying possible suppression of malignant phenotype and secondly, we analysed DNAs from 46 primary breast cancers for loss of constitutive heterozygosity (LOH) using 24 poly-morphic microsatellite markers. The chromosome transfer resulted in loss of tumorigenicity and reversion of other neoplastic properties of the microcell hybrids. Polymorphism analysis of single hybrids revealed that they harbored only a small donor chromosome fragment defined by the marker D6S310 (6q23.3-q25) and flanked by D6S292 and D6S311. The LOH data suggest that four tumor suppressor gene loci mapped to the central and distal portion of 6q may be independently deleted in breast cancer. One of these regions corresponds to the region identified by chromosome transfer.

Detection of cell-free nucleic acids in bronchial lavage fluid supernatants from patients with lung cancer.

The aim of this study was to determine whether nucleic acids are detectable in cell-free bronchial lavage supernatants, and whether it is possible to find alterations in this DNA and RNA of genes known to be present in lung tumour cells. DNA was isolated from cell-free lavage supernatants from 30 and RNA from 25 lung cancer patients. The DNA was examined for microsatellite alterations (MA) and the RNA analysed for the expression of seven tumour-associated genes. Intact DNA and mRNA could be isolated from all cell-free bronchial lavage supernatants. MA were found in lavage supernatants of 12/30 patients and in lavage cells of 6/30 patients. Altogether alterations were found in 14/30 patients. Analyses of tumour-associated gene expression showed positive results, with at least one marker in the lavage supernatants of all 25 patients. Thus, we could demonstrate, for the first time, that it is possible to isolate intact DNA and RNA from cell-free bronchial lavage supernatants. Their quantity and quality is sufficient for further amplification by polymerase chain reaction (PCR)/reverse transcriptase (RT)-PCR. Altogether, tumour-associated changes were detected in DNA samples from 47% of the patients and in RNA samples from all of the patients analysed.

Membrane transport in multidrug resistance, development, and disease. AACR special conference in cancer research.

The main goal of this meeting was to provide the scientists and clinicians active in this field with a comprehensive overview of the progress that has been made. The meeting was a forum in which new advances in membrane transport were discussed in depth and which gave new impulses for clinical applied research. Again, the importance of intensive cooperation between basic research and clinical use became evident during this symposium.

BRCA1 mutation update and analysis.

The discovery of the BRCA1 gene involved in the development of human hereditary breast cancer led to extensive international efforts to identify the mutations leading to the disease. The new listing covers 127 mutations published in the indicated papers before 30 April 1996; 55% of the mutations are localized in exon 11, followed by exons 2 (5.5%), 5 and 16 (4.7% each).

Identification of a recurrent BRCA1 mutation in German breast-cancer and/or ovarian-cancer families.

Specific BRCA1 mutations have been reported to be common within particular populations. We have investigated German breast- and/or ovarian-cancer families and detected a recurrent carboxy-terminal BRCA1 mutation, 5622C > T, using PCR-based restriction assay and haplotype analysis. Unrelated families carrying this BRCA1 mutation shared two different disease-associated haplotypes, indicating two independent mutation events.

Detection of amplifiable messenger RNA in the serum of patients with lung cancer.

Recently, in addition to the detection of circulating tumor cells in peripheral blood of patients with solid tumors, the presence of free circulating nucleic acids in the plasma and serum has also been described. We have focused on the possibility of isolating and amplifying intact extracellular, tumor-related mRNA from the plasma/serum of patients with lung cancer. For this purpose, we established several RT-PCR-based amplification systems for the detection of a panel of five different genes. The expression of these genes was either shown to be restricted to lung tissue or associated with malignancy. We examined two small groups of 18 patients with lung cancer before and during chemotherapy, respectively. The message for beta-actin (control for integrity of the RNA) was detected in all of the analyzed sera from the control group and patients with lung cancer. Analysis of CK-19 expression was positive in the majority of tumor patients, but positive results were also shown in all of the control sera. The expression of MAGE-2 and TTF-1 genes was not observed in any of the patients in either the lymphocyte preparations or serum samples. Expression of the PGP 9.5 gene was observed in the cells of all 18 patients, but mRNA in the serum was only detectable in one case. The hnRNP-B1 mRNA was detectable in 14/18 sera, and Her2/neu-specific mRNA could be amplified from the serum of 7/18 patients. Combining the last two markers, we were able to detect all patients with a malignant lung tumor.

Detection of tumor-specific mRNA in cell-free bronchial lavage supernatant in patients with lung cancer.

Bronchoscopy is a standard procedure in the workup of patients with suspicious pulmonary lesions. We wondered whether it is possible to isolate malignancy-associated mRNA from cell-free lavage supernatant. Extracellular mRNA from cell-free lavage supernatant of 25 patients with lung cancer (23 with non-small cell lung cancer, 2 with small cell lung cancer) was isolated, reverse-transcribed, and amplified by reverse transcriptase polymerase chain reaction. The quantity and quality of the isolated RNA were checked after cDNA synthesis by amplification with beta-actin-specific primers. Afterwards, a panel of eight genes known to be expressed in lung tumors was used for the detection of tumor-associated mRNA expression in lavage supernatant and serum. mRNA coding for beta-actin could be isolated from lavage supernatant of all 25 patients. In addition, the expression of at least one tumor-associated gene was detectable in all patients. These results show that intact mRNA can be isolated from cell-free lavage supernatant and that its quantity and quality are sufficient for the detection of tumor-associated gene expression alterations. This may open new possibilities for the diagnosis of lung cancer.

Detection of microsatellite alterations in the DNA isolated from tumor cells and from plasma DNA of patients with lung cancer.

In this paper, we show that the same panel of three microsatellite markers is useful for the detection of alterations in the DNA of tumor cells and plasma from patients diagnosed with SCLC and NSCLC. In 31% of the SCLC patients, we detected a microsatellite alteration(s) or LOH in at least one locus. In the group of patients diagnosed with NSCLC, a microsatellite alteration or LOH was detected in at least one locus in 33% of the patients. In all but 2 patients, the identical alteration observed in the DNA from tumor cells was also detected in the DNA isolated from blood plasma. This work confirms the results described by other groups and it extends the diagnostic possibilities of finding tumor cell-specific DNA alterations also in the DNA freely circulating in plasma and serum of patients with cancer.

Estradiol receptor and prognostic parameters of human breast cancer.

Estradiol receptors are regarded to predict a likely success of hormonal therapeutic efforts and the prognosis of breast cancer patients. But today its prognostic importance is controversial, discussed as either reflecting intrinsic property of the tumor tissue or better therapeutic accessibility of receptor positive tumors. Moreover, the most important clinical prognosticators--tumor size and axillary lymph node involvement do not seem to be related to the estradiol receptor status. In our investigation, the length of disease free interval is similar in estradiol receptor positive and negative patients and in all sites of distant metastases, but it is significantly reduced if more than 4 axillary lymph nodes are involved. Post recurrence survival is significantly longer in estradiol receptor positive than negative patients and also in patients treated by tamoxifen containing therapies. Its length is independent of the number of axillary lymph node metastases and the type of distant metastases, with a tendency to be longer in estradiol receptor positive than negative patients. In addition, the overall survival is longer for estradiol receptor positive than negative patients and becomes reduced with more than 4 axillary lymph node metastases. Frequency of deaths in estradiol receptor positive patients is half that of negative subjects. Furthermore, the length of overall survival is independent on the type of distant metastases, with tendency to be longer in estradiol receptor positive than negative patients. Longest overall survival could be observed for estradiol receptor positive patients who got therapy regimens containing tamoxifen. The weak prognostic advantages of estradiol receptor positive patients are interpreted by estradiol receptors as intrinsic parameters of breast cancer tissue characterizing more its biological behavior than therapeutic accessibility.

Activated fos oncogene in rat embryo fibroblasts transformed by ras and myc oncogenes.

Rat embryo fibroblasts (REF) were transformed by simultaneous gene transfer of the complementary oncogenes ras and myc using the calcium phosphate coprecipitation method. Cell lines derived from transformation foci expressed in addition to ras and myc cellular oncogene fos while normal REF did not express ras, myc and fos according to the hybridization methods used. The transformed cell lines produced colonies in soft agar and tumors in newborn syngeneic rats. From one tumor a cell line was established which was characterized by a high level of fos gene expression.

Steroid hormone receptors and antineoplastic chemotherapy in human breast cancer.

Several clinical and experimental investigations suggest that the action of antineoplastic chemotherapy in premenopausal women influences the menopause. Such hormonal reactions are mediated via specific steroid hormone receptors. Therefore, connections between hormone receptors and antineoplastic chemotherapy can be assumed making possible to predict success of chemotherapy on the basis of receptor status. Nevertheless, clinical experiences and animal and cell culture experiments yielded controversial results. This was related to the predictive value of receptor status as well as to the benefits of combined hormone and chemotherapy treatments in concurrent or sequential form. It is undeniable that a displacing of steroidal ligand from its receptors by the usual antineoplastic drugs does not occur. Furthermore, the receptor levels remain unchanged after a treatment with antineoplastic drugs. Thus, the mechanism of action of chemotherapeutic drugs is not related directly to the presence or absence of steroid hormone receptors. Despite this fact the receptor status in chemotherapeutic regimes seems to be helpful to define low or high risk patients. Influences on the ER de-novo-synthesis, actions related to parameters representing reduced tumor growth rates, down-regulation of the receptor gene expression or via receptor mediated hormonal actions to other genes, like the apoptosis-related gene bcl-2, are thought to be possible mechanisms of action of antineoplastic drugs on steroid hormone receptors. Future investigations should monitor the ratios between exon lacking receptor variants and the wild-type receptor during chemotherapy or the control of a ligand uptake during chemotherapy by means of the positron emission tomography.

Estradiol receptors and metastasis in human breast cancer.

The localization and extent of metastasis determined the prognosis of breast cancer in a decisive manner. Thereby axillary lymph node involvement represents one of the most important prognostic indicators. The estradiol receptor status is also attributed some prognostic importance. There might therefore be relations between these prognostic factors. However, the majority of investigators could not find a correlation between the extent and timing of regional lymph node involvement and estradiol receptor status. In contrast, there are numerous findings which confirm correlations between estradiol receptors and the localization, extent, and timing of distant metastasis. The findings obtained in more recent years have been collected and discussed in relation to events included in the process of metastasis such as release of proteases and existence of receptor variants.

Steroid hormone receptors related to parameters characterizing the biology of human breast cancer.

Steroid hormone receptors are important parameters to characterize breast tumors. Thus, it is important to evaluate their relationships with factors such as histological type and size of the tumor, axillary lymph node invasion and distant spread, age and menopausal status of the patients, and parameters of tumor differentiation. The receptor levels observed vary within wide ranges. Therefore, statistically significant differences between different groups of parameters are seldom found. A significant dependence on receptor levels has been observed only for patient age and menopausal status. The parameters clinical stage, tumor size, tumor histology, metastatic involvement and histopathologic grading showed no statistically significant relation with receptor levels. Nevertheless, a relationship exists between all parameters mentioned and the frequency of a positive receptor status. Consequently, receptor status can contribute to define the biological behavior of the disease in specific groups of patients. In individual cases other parameters than the biochemical evidence of steroid receptor binding seem to be more important. We found that data derived from DNA flow cytometric measurements allowed a better recognition of tumor aggressiveness than ER status. In axillary lymph node metastases we usually observed higher receptor levels than in primary tumors, but we did not find an age dependency of ER levels in these metastases.

Polyomavirus infection in hamsters and trichoepitheliomas/cutaneous adnexal tumours.

Multiple skin nodules, with histological features of adnexal tumours consistent with trichoepithelioma, were observed on the head and trunk of Syrian hamsters. Skin biopsies from 20 hamsters from five different colonies were affected, and two of the affected hamsters also had lymphoma. Two owners reported that 16 of 70 hamsters and 50 of 100 hamsters in their colonies had similar skin lesions. These tumours have previously been associated in laboratory colonies with hamster polyomavirus (HaPV) infection. Examination of skin tissues by electron microscopy failed to reveal intranuclear virus particles. Using recombinant major capsid protein VP1 of HaPV, VP1-specific antibodies were detected in sera from 12 of 12 affected hamsters and in four of four unaffected in-contact hamsters, by ELISA. The ELISA data were verified by immunoblot analysis. Eleven of 13 serum samples contained antibodies which reacted with at least one recombinant structural HaPV protein (VP2), including samples from three in-contact unaffected hamsters. Nine of the 11 anti-VP2-positive samples also reacted with recombinant VP3 of HaPV, and six reacted with VP1. Amplification by PCR and sequencing detected VP1 -encoding sequences showing a high degree of homology with HaPV. The findings suggest a possible infection by HaPV or a HaPV-like virus and it is likely that such an infection was enzootic within the affected colonies.

Amplification of oncogenes and disease prognosis.

Amplification is one mechanism for activation of oncogenes and results in an excess of DNA template, which can lead to overproduction of oncogene-specific RNA and protein. Amplification of oncogenes has been observed in different tumor tissues. In certain cases amplification and overexpression of particular oncogenes have been correlated with tumor progression and clinical behavior. The best example is neuroblastoma in which the N-myc oncogene frequently is found to be amplified. Over 1,000 patients with breast cancer have been studied for amplification of the c-erbB-2 oncogene until now. The evidence from the studies that amplification of c-erbB-2 is correlated with poor prognosis is in our opinion not convincing. More and more investigations about oncogenes and disease prognosis will take place rather at the protein level than at the DNA level.

In vitro transformation of rat cells by 3-methylcholanthrene: activation of the ras oncogene.

Rat cells of the established, immortalized line rat-2 were treated with the polycyclic aromatic hydrocarbon 3-methylcholanthrene. No characteristic morphological transformation occurred during three weeks after treatment. However, the carcinogen-treated cells formed colonies in soft agar. Cell lines established from single soft agar colonies were characterized by an increased proliferation rate, an enhanced colony formation in soft agar and an increased expression of the Ha-ras oncogene.

Generation of lymphoma-type variant hamster polyomavirus genomes in hamsters susceptible to lymphoma induction.

The hamster polyomavirus (HaPV) induces either hair follicle epitheliomas or lymphomas in either Z3 or HaP respectively. Syrian hamsters. In the lymphomas specifically deleted "lymphoma-type" (lt) HaPV genomes are accumulated. In the present study the temporal pattern of generation of HaPV (lt) DNA was investigated in context of the development of lymphomas in neonatally infected HaP hamsters. The generation of HaPV (lt) DNA was first detectable during the postnatal phase of high level replication of viral DNA in hemopoietic organs (at 7 days post infection), thus clearly preceding the development of overt lymphoma. A variety of HaPV (lt) DNA species is generated in lymphoid cells, but usually only one of them is accumulated to high amounts in lymphoma cells. Furthermore, the pattern of HaPV (lt) and wild-type (wt) DNA was studied in normal and tumor tissues of tumor-bearing hamsters as well as in tumor-free hamsters. In tumor-bearing hamsters predominantly HaPV (lt) DNA species were found in the infected tissues, while HaPV (wt) DNA was detected rarely and only in tumor-free tissues. In contrast, in tissues of tumor-free hamsters HaPV (wt) DNA prevailed over HaPV (lt) DNA species.

Transformation of rodent immortalized and embryo cells by oncogenes. I. Mouse (NIH 3T3) and rat (FR 3T3) immortalized fibroblasts.

Immortalized mouse NIH 3T3 cells were transformed by gene transfer of DNA isolated from a human bladder tumor cell line and plasmids containing an activated human Ha-ras oncogene insert. For gene transfer the calcium-phosphate co-precipitation method was used. Transformation was evaluated by morphological focus formation, growth in soft agar and tumor development in nude mice. In addition, immortalized rat FR 3T3 cells were transformed by Ha-ras, too. The co-transfer of ras and myc oncogenes did not enhance focus formation in FR 3T3 cells.