Modified differential display technique that eliminates radioactivity and decreases screening time.

Several techniques are available that detect variations in gene expression between cellular populations. These include subtractive hybridization (SH), differential colony hybridization (DCH) and mRNA differential display, all based on the analysis of mRNA. The first two techniques, however, are limited because they require large amounts of mRNA for SH or several rounds of screening for DCH. Differential display overcomes both of these limitations. However, the conventional differential display technique is plagued by false positives and is labor intensive. The identification of genes that are truly differentially expressed, therefore, becomes a formidable task. We describe a modified differential display technique that overcomes the limitations of the conventional technique. This new technique eliminates a source of false positives, decreases the time required to screen a set of primers and reduces the use of radioactivity.

A high-fat diet does not influence the growth of the Dunning R3327-H transplantable prostate adenocarcinoma.

BACKGROUND: The etiology of prostate cancer is currently a mystery. Several epidemiological studies suggest a link between dietary fat and prostate cancer. In vitro and in vivo studies support this evidence. Using the Dunning model of rat prostate cancer we hypothesized that a high-fat diet (20%) would increase the growth of the R3327-H tumor. MATERIALS AND METHODS: R3327-H tumors were implanted subcutaneously into male Copenhagen rats which were fed diets with 5 or 20% total fat. Tumors were allowed to grow for 16 weeks; they were then excised and weighed. The initial and final weights of the rats were also recorded. RESULTS: Statistical analysis revealed the level of dietary fat was a positive predictor of weight gain (p < 0.01). No effect on tumor growth was seen when compared to dietary fat, fiber type, or the interaction of fat and fiber. DISCUSSION: Growth of the R3327-H tumor, when implanted subcutaneously, is not affected by the level of dietary fat.

Examination of the DNA methylation properties in nontumorigenic and tumorigenic breast epithelial cell lines.

Molecular changes in the progressive state of tumorigenesis often include altered patterns of DNA methylation. Utilizing a series of breast epithelial cell lines, the overall 5-methylcytosine content in genomic DNA demonstrated an overall decrease when comparing two malignant cell lines (MCF-7 and T47D) with a mortal cell line (MCF 1 2M) and several derivative cell lines of the immortalized MCF10 cultures (MCF10A,-2A, -5A, A1neoT2, and 139B6). Further investigation on the methylation status of these cells lines indicated no difference in DNA methyltransferase activity, both at a protein and mRNA levels, in the nontumorigenic cell lines examined while activity was 3-10 fold higher in the tumorigenic lines (MCF7, T47D, SkBr3, MB-MDA-231, -468). Examination of the CpG island in the 5' promoter region of the estrogen receptor gene indicates that this region is unmethylated in the mortal and immortal nontumorigenic lines as well as the tumorigenic lines examined, with the exception of the estrogen receptor negative breast cell line MB-MDA-468 which appears to be partially methylated at this site. These results indicate methylation of this CpG island does not account for the inactivation of the estrogen receptor gene in immortalized nontumorigenic breast cells, suggesting another mechanism of transcriptional inactivation of ER in this environment.

The effect of castanospermine on the metastatic properties of prostate cancer cells.

BACKGROUND: Most deaths from prostate cancer result from the metastatic spread of the disease. Castanospermine has been shown to inhibit tumor growth and metastasis in mouse and rat models. We hypothesized that castanospermine might inhibit metastasis in the Dunning model of rat prostate adenocarcinoma by interfering with the metastatic properties of tumor cells. MATERIALS AND METHODS: We examined the cytotoxicity of castanospermine toward the metastatic MAT-LyLu and nonmetastatic AT. 1 cell lines and its effects on cell motility and adhesion to endothelial cells. We assessed castanospermine's effects on in vivo metastasis in Copenhagen rats. RESULTS: Castanospermine was not cytotoxic toward the MAT-LyLu and AT. 1 cell lines at concentrations through 10 micrograms/mL, nor did it significantly affect cell motility, adhesion to endothelial cells, or in vivo metastasis. CONCLUSIONS: Within the Dunning model, castanospermine did not appear to significantly affect cell characteristics related to metastatic potential.

Modeling signal transduction in normal and cancer cells using complex adaptive systems.

As a first approximation, organisms can be defined by the complement of cell types that they possess. Each cell type is defined by its specific collection of signal transduction pathways. While many pathways are common to most cell types (e.g. glycolysis), others are specific to a particular cell type and serve to characterized that cell. Many diseases, including cancer, are characterized by aberrations in general and specific signal-transduction pathways. These pathways are generally intricate and not easily modeled. The formalism of complex adaptive system theory, however, provides the tools by which these pathways can be investigated. By modeling signal-transduction pathways from the viewpoint of complex adaptive systems, a deeper understanding of their intricacies may result. This could eventually lead to novel methods of therapeutic intervention in diseases that arise from aberrant signal transduction.

Development of doxorubicin resistant rat prostate cancer cell lines.

BACKGROUND: One possible mechanism of chemotherapeutic resistance in patients with metastatic prostate cancer is the overexpression of P-glycoprotein. Additional tumor models are necessary to study this phenomenon. MATERIALS AND METHODS: Doxorubicin resistant rat prostate cancer cell lines were developed by increasing doxorubicin levels in cell culture. RESULTS: The MDR lines (AT3B-1, AT3B-2, MLLB-1, and MLLB-2) were more resistant to vinblastine compared to controls. When P-glycoprotein was blocked, the AT3 MDR lines demonstrated efflux activity. Injection of AT3 MDR lines into rats followed by doxorubicin treatment produced larger tumors compared to the parental controls. CONCLUSIONS: MDR rat prostate cancer cells were developed. AT3B-1 and AT3B-2 cell lines have drug efflux pump ability, whereas the MLLB-1 and MLLB-2 may not, suggesting alternative key mechanisms other than P-glycoprotein overexpression. These new cell lines are being used to study chemotherapy resistance in prostate cancer.

Cancer as a complex adaptive system.

The second leading cause of death in the USA is cancer. Institutions worldwide are devoting significant resources to the treatment of cancer, and the elucidation of the disease pathway. While great progress has been made in understanding and treating carcinogenesis, many aspects of the disease remain intractable. Throughout the history of science many other disciplines--astronomy, particle physics, etc.--have been advanced when the fundamental ideas governing the discipline were redefined. These redefinitions are often termed 'paradigm shifts'. The new sciences of chaos theory and complexity have led to paradigm shifts in many unrelated disciplines such as economics, meteorology and seismology. Our current understanding of carcinogenesis has resulted from a conventional view of the disease process. In this perception, the mutation of a gene, or several genes, leads to cancer. Applying the formalism of chaos theory and complexity to carcinogenesis, however, leads to a different perception of the disease. If we look closer, cancer can be viewed as a complex adaptive system. Redefining our perception of cancer may lead to a deeper understanding of the disease, and possibly result in novel methods of therapeutic intervention.