Establishment of New Genetic Markers and Methods for Sex Determination of Mouse and Human Cells using Polymerase Chain Reactions and Crude DNA Samples.

Background: The currently available methods for sexing human or mouse cells have weaknesses. Therefore, it is necessary to establish new methods. Methods: We used bioinformatics approach to identify genes that have alleles on both the X and Y chromosomes of mouse and human genomes and have a region showing a significant difference between the X and Y alleles. We then used polymerase chain reactions (PCR) followed by visualization of the PCR amplicons in agarose gels to establish these genomic regions as genetic sex markers. Results: Our bioinformatics analyses identified eight mouse sex markers and 56 human sex markers that are new, i.e. are previously unreported. Six of the eight mouse markers and 14 of the 56 human markers were verified using PCR and ensuing visualization of the PCR amplicons in agarose gels. Most of the tested and untested sex markers possess significant differences in the molecular weight between the X- and Y-derived PCR amplicons and are thus much better than most, if not all, previously-reported genetic sex markers. We also established several simple and essentially cost-free methods for extraction of crude genomic DNA from cultured cells, blood samples, and tissues that could be used as template for PCR amplification. Conclusion: We have established new sex genetic markers and methods for extracting genomic DNA and for sexing human and mouse cells. Our work may also lend some methodological strategies to the identification of new genetic sex markers for other organismal species.

About three-fourths of mouse proteins unexpectedly appear at a low position of SDS-PAGE, often as additional isoforms, questioning whether all protein isoforms have been eliminated in gene-knockout cells or organisms.

Most genes in evolutionarily complex genomes are expressed to multiple protein isoforms, but there is not yet any simple high-throughput approach to identify these isoforms. Using an oversimplified top-down LC-MS/MS strategy, we detected, around the 26-kD position of SDS-PAGE, proteins produced from 782 genes in a Cdk4-/- mouse embryonic fibroblast cell line. Interestingly, only 213 (27.24%, about one-fourth) of these 782 genes have their proteins with a theoretical molecular mass (TMM) 10% smaller or larger than 26 kD, that is, between 23 and 29 kD, the range set as allowed variation in SDS-PAGE. These 213 proteins are considered as the wild type (WT). The remaining three-fourths includes proteins from 66 (9.44%) genes with a TMM smaller than 23 kD and proteins from 503 (64.32%, nearly two-thirds) genes with a TMM larger than 29 kD; these proteins are categorized into a larger-group or a smaller-group, respectively, for their appearance at a higher or lower position of SDS-PAGE. For instance, at this 26-kD position we detected proteins from the Rps27a, Snrpf, Hist1h4a, and Rps25 genes whose proteins' TMM is 8.6, 9.7, 11.4, and 13.7 kD, respectively, and detected proteins from the Plelc1 and Prkdc genes, whose largest isoform is 533.9 and 471.1 kD, respectively. We extrapolate that many of those proteins migrating unexpectedly in SDS-PAGE may be isoforms besides the WT protein. Moreover, we also detected a Cdk4 protein in this Cdk4-/- cell line, thus wondering whether some of other gene-knockout cells or organisms show similar incompleteness of the knockout.

Gene expression profiles in primary pancreatic tumors and metastatic lesions of Ela-c-myc transgenic mice.

BACKGROUND: Pancreatic carcinoma usually is a fatal disease with no cure, mainly due to its invasion and metastasis prior to diagnosis. We analyzed the gene expression profiles of paired primary pancreatic tumors and metastatic lesions from Ela-c-myc transgenic mice in order to identify genes that may be involved in the pancreatic cancer progression. Differentially expressed selected genes were verified by semi-quantitative and quantitative RT-PCR. To further evaluate the relevance of some of the selected differentially expressed genes, we investigated their expression pattern in human pancreatic cancer cell lines with high and low metastatic potentials. RESULTS: Data indicate that genes involved in posttranscriptional regulation were a major functional category of upregulated genes in both primary pancreatic tumors (PT) and liver metastatic lesions (LM) compared to normal pancreas (NP). In particular, differential expression for splicing factors, RNA binding/pre-mRNA processing factors and spliceosome related genes were observed, indicating that RNA processing and editing related events may play critical roles in pancreatic tumor development and progression. High expression of insulin growth factor binding protein-1 (Igfbp1) and Serine proteinase inhibitor A1 (Serpina1), and low levels or absence of Wt1 gene expression were exclusive to liver metastatic lesion samples. CONCLUSION: We identified Igfbp1, Serpina1 and Wt1 genes that are likely to be clinically useful biomarkers for prognostic or therapeutic purposes in metastatic pancreatic cancer, particularly in pancreatic cancer where c-Myc is overexpressed.

Therapeutic intervention of experimental breast cancer bone metastasis by indole-3-carbinol in SCID-human mouse model.

Several lines of experimental evidence have suggested that chemokine receptor CXCR4, a metastasis-promoting molecule, may play important roles in breast cancer bone metastasis. There is emerging evidence linking CXCR4 to matrix metalloproteinases (MMP) as well as their regulator nuclear factor-kappaB (NF-kappaB), a key transcription factor, which is known to activate metastasis-promoting molecules for many types of malignancies, including breast cancer. A recent study also showed that promoter region of CXCR4 has several NF-kappaB-binding sites, suggesting that there may be a cross-talk between CXCR4 and NF-kappaB. We have shown previously that indole-3-carbinol (I3C), a natural compound present in vegetables of the genus Brassica, can inhibit NF-kappaB in breast cancer cells. However, there are no reports in the literature showing any effect of I3C on CXCR4 expression in vitro and in vivo. We therefore examined whether I3C could inhibit bone metastasis of breast cancer by inhibiting CXCR4 and MMP-9 expression mediated via the inhibition of the NF-kappaB signaling pathway. Here, we have modified the severe combined immunodeficient (SCID)-human mouse model of experimental bone metastasis for use with the MDA-MB-231 breast cancer cell line. In this animal model, we found that I3C significantly inhibited MDA-MB-231 bone tumor growth, and our results were correlated with the down-regulation of NF-kappaB. Moreover, we found that I3C significantly inhibited the expression of multiple genes involved in the control of metastasis and invasion in vitro and in vivo, especially the expression of CXCR4 and MMP-9 along with pro-MMP-9, with concomitant decrease in Bcl-2 and increase in the proapoptotic protein Bax. From these results, we conclude that the CXCR4/NF-kappaB pathway is critical during I3C-induced inhibition of experimental breast cancer bone metastasis. These results also suggest that I3C could be a promising agent for the prevention and/or treatment of breast cancer bone metastasis in the future.

Roles of androgens in the development, growth, and carcinogenesis of the mammary gland.

Androgens influence the development and growth of the mammary gland in women. Treatment of animals and cultured cells with androgens has either inhibitory or stimulatory effects on the proliferation of mammary epithelia and cancer cells; the mechanisms for these dual functions are still not very clear and are discussed in this review. Epidemiological data suggest that, similar to increased estrogens, elevated androgens in serum may be associated with the development of breast cancer. Experiments in rodents have also shown that simultaneous treatment of androgen and estrogen synergizes for mammary gland carcinogenesis. Similar synergistic effects of both hormones have been observed for carcinogenesis of the uterine myometrium of female animals and for carcinogenesis of the prostate and deferens of males. There are also clinical and experimental indications for a possible association of elevated levels of both androgens and estrogens with the development of ovarian and endometrial cancers. A hypothesis is thus proposed that concomitant elevation in both androgens and estrogens may confer a greater risk for tumorigenesis of the mammary gland, and probably other female reproductive tissues than an elevation of each hormone alone.