The present study describes the development and optimization of a cell-based reporter assay for the determination of androgen bioactivity levels in human serum samples. Towards this end, human embryonic kidney (HEK) 293 cells were cotransfected with two plasmids, one encoding the mouse mammary tumor virus (MMTV)-driven luciferase reporter gene and the other the SV40 promoter-driven human androgen receptor (AR), and a stable cell line, expressing human AR and androgen-responsive luciferase was established by antibiotic selection. RT-PCR confirmed proper transcription and stable integration of AR in the cell line. On stimulation of the cells with testosterone (T) for 24h, luciferase activity was increased in dose-dependent fashion up to 15-fold, with the minimum effective concentration of T being 0.03 nmol/l. T-induced reporter gene expression of the cells was inhibited by the anti-androgens cyproterone acetate and hydroxyflutamide. Upon stimulation with non-androgenic steroids like estradiol, progesterone, dexamethasone and cortisol, the cells showed only marginal activity except for a weak glucocorticoid effect at high concentration. The cells also responded well to various chemicals (mostly pesticides, their metabolites and common industrial chemicals) with known androgenic activity. The cell line was further optimized by measuring the levels of androgens from male and female serum samples. Our data indicated that the cells can be used to estimate androgen bioactivity in serum of females suffering from polycystic ovary syndrome (PCOS) and males of different age groups. In conclusion, we demonstrate that the bioassay based on this cell line provides a reliable method to determine serum levels of androgen bioactivity from biological samples even at the low concentrations present in female circulation. The 96-well plate format makes the assay suitable for high throughput measurements.

Rat-1 cells are used in many studies on transformation, cell cycle, and apoptosis. Whereas UV treatment of Rat-1 cells results in apoptosis, X-ray treatment does not induce either apoptosis or a cell cycle block. X-ray treatment of Rat-1 cells results in both an increase of p53 protein and expression of the p53-inducible gene MDM2 but not the protein or mRNA of the p53-inducible p21(WAF1/CIP1) gene, which in other cells plays an important role in p53-mediated cell cycle block. The lack of p21(WAF1/CIP1) expression appears to be the result of hypermethylation of the p21(WAF1/CIP1) promoter region, as p21(WAF1/CIP1) protein expression could be induced by growth of Rat-1 cells in the presence of 5-aza-2-deoxycytidine. Furthermore, sequence analysis of bisulfite-treated DNA demonstrated extensive methylation of cytosine residues in CpG dinucleotides in a CpG-rich island in the promoter region of the p21(WAF1/CIP1) gene. Stable X-ray-induced p53-dependent p21(WAF1/CIP1) expression and cell cycle block were restored to a Rat-1 clone after transfection with a P1 artificial chromosome (PAC) DNA clone containing a rat genomic copy of the p21(WAF1/CIP1) gene. The absence of expression of the p21(WAF1/CIP1) gene may contribute to the suitability of Rat-1 cells for transformation, cell cycle, and apoptosis studies.

Using a polymerase chain reaction (PCR)-based strategy that distinguishes functional intron-containing genes from pseudogenes, the chromosomal location of the human intron-containing L35a ribosomal protein gene (rpL35a, HGMW-approved symbol RPL35A) was previously deduced to be onchromosome 18 using DNAs from a panel of human-rodent somatic cell hybrids. The inability to detect rpL35a in a human-hamster somatic cell hybrid containing only human chromosome 18 led to a reinvestigation of the chromosomal location of the human rpL35a gene. A clone containing intron 2 of rpL35a was isolated, sequenced, and used to isolate a P1 clone containing the human intron-containing rpL35a gene. By fluorescence in situ hybridization (FISH) analysis with the P1 clone as a probe, the rpL35a gene was located on chromosome band 3q29-qter. This location was confirmed by positive PCR analysis of a human-hamster somatic cell hybrid containing only human chromosome 3. The problem of using only human-rodent somatic cell hybrids for chromosome location is discussed.

Integrated viral sequences and adjacent cellular sequences from the polyoma virus (Py)-transformed 53-Rat and 82-Rat cell lines which contain two and three partial early regions respectively, each in a single viral insert, have been molecularly cloned. Each of the cloned partial early regions have been subcloned and assessed with regard to their transcription, translation products (T antigens, T Ags) and biological activity including their transforming ability. The 53-Rat 5.3 kb EcoRI fragment is an intact Py EcoRI linear genome (derived from within the tandem duplicated sequences) which transforms rat cells with high efficiency and produces infectious virus when circularized and transfected into mouse cells. The 82-Rat cell line expresses three novel T Ag species of 63K, 40K and 32K in addition to the Py middle and small T Ags. The 63K protein was found to be a truncated form of large T Ag produced as the result of an addition/deletion in early region B sequences unique to large T Ag. The 40K and 32K proteins are hybrid viral-cellular middle and large T Ags respectively, which are expressed from early region A that has been truncated by recombination with rat cellular DNA. Differences in the nuclear and cytoplasmic location of the different 82-Rat early region RNAs are due to RNA stability and/or transport from the nucleus to the cytoplasm most likely as a result of different cellular sequences at their 3' ends. Finally no common structural feature or sequence specificity was observed at the virus-host DNA joins of the two cell lines.