Louis A. Matej

Regulation of sex hormone-binding globulin production by growth factors.

Sex hormone-binding globulin (SHBG) is a glycoprotein whose production has been demonstrated to be regulated by both sex steroids, as well as by thyroid hormone and peptide hormones such as insulin. However, none of these regulatory factors would explain the marked decrease in serum SHBG seen throughout the prepubertal and pubertal time period in both boys and girls. Furthermore, current in vitro data show that both androgens and estrogens can stimulate SHBG production by the human hepatoblastoma cell line Hep G2; yet, in vivo androgens appear to suppress SHBG levels, while estrogens are associated with elevated levels. This study was undertaken to determine possible mechanisms to explain this phenomenon. Hep G2 cell cultures were incubated with insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), or dehydroepiandrosterone (DHEA). Significant decreases in the level of SHBG in the culture medium relative to control cultures occurred for each of the growth factors (P less than .01), whereas an increase in SHBG levels was observed in the medium of DHEA-treated cells. When cells were coincubated with IGF-I and thyroxine (T4), which alone stimulates SHBG production both in vivo and in vitro, the SHBG response to T4 was blunted. These results suggest that growth factors, as well as insulin, may be important determinants in SHBG production.

Acclimation of a non-indigenous sub-Arctic population: seasonal variation in thyroid function in interior Alaska

Total, as well as free, T4 and T3 levels were obtained over four seasons for young male infantry soldiers assigned to interior Alaska. Significant seasonal variations were found in both T3 and T4. Total T4 and T3 levels were highest in winter, while free T4 and T3 levels were highest in early spring. Correlations with melatonin levels from a concurrent study showed an association between late day (17.00) mean spot melatonin levels during the preceding summer and T3 levels in winter and spring. Differences in seasonal T4 and T3 levels between indigenous and newly arrived people in the sub-Arctic may be related not only to cold acclimation but also to light.

Sex hormone binding globulin mRNA in human breast cancer: Detection in cell lines and tumor samples

Sex hormone binding globulin (SHBG) is a high affinity binding protein for estrogens and androgens. SHBG has been found in breast tissue and cell lines through immunostaining. The goal of this series of experiments was to determine whether mRNA for SHBG is expressed in breast cancer cell lines and tumor tissue. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect SHBG and beta-2 microglobulin (control for tissue extractions). Three breast cancer cell lines, ZR-75-1, MCF-7, and MDA-MB-231 and 56 breast tissue samples were collected and analysed for SHBG mRNA expression. mRNA was successfully extracted from 30 of these breast tissue samples. SHBG mRNA was detected in ZR-75-1, MCF-7 and MDA-MB-231 cells, and in 11 of the breast tissue samples. Two PCR products were routinely amplified from the breast cancer cell line RNA, one at approximately 500 bp and another at approximately 300 bp. The DNA sequence of the 300 bp PCR produce was consistent with alternate splicing of the SHBG mRNA, where exon 7 is deleted, and is accompanied by a point deletion at the beginning of exon 8. SHBG protein production from the three breast cancer cell lines was detected by immunoprecipitation using an affinity purified SHBG antibody. SHBG mRNA was found in 11 of 30 samples of breast tissue. Some samples expressed only the 500 bp or the 300 bp PCR product, whereas others expressed both PCR products. The presence of SHBG mRNA in these samples was not associated with either the presence or absence of steroid receptors. SHBG mRNA is thus expressed in breast cancer cell lines, and in some breast tissue samples.

Seminal Fluid Androgen Binding Protein

The following study was undertaken to determine the presence of an androgen binding protein (ABP) in the ejaculate. Seminal fluid was obtained from ten fertile subjects. Sperm counts ranged from 20 times 106/cc to 80 times 106/cc. Each sample was preincubated with tritiated dihydrotestosterone (DHT) and examined by polyacrylamide gel electrophoresis (PAGE). Second, after preincubation of the samples with tritiated DHT, the samples were mixed with Concanavalin‐A bound to sepharose‐D (C‐S) and the supernate separated and counted. Finally, saturation analysis of the samples using dextran‐coated charcoal for separation was performed and Scatchard analysis was used to determine the concentration of ABP present.

Expression and characterization of novel thrombospondin 1 type I repeat fusion proteins.

Thrombospondin (TSP)1 is a trimeric extracellular matrix protein that is held together by two cysteine residues. It is one of five TSP proteins that have been described to date with almost a universal heparin binding capability (TSP5 being the exception). The existence of two conformationally distinct structures in the TSP family (trimers and pentamers) prompted us to investigate the contribution of TSP1 trimeric structure to its inhibitory role in angiogenesis. We expressed full-length recombinant human TSP1, its type I repeats, and murine TSP3 in a human embryonic kidney cell line and evaluated their effect on human dermal microvascular endothelial cell (HMVEC) proliferation and sprouting into tube-like structures in vitro. Additionally, two chimaeric molecules were constructed so that the type I repeats of TSP1 were expressed as either dimers (TSP1-Ig chimaera) or pentamers (TSP1-TSP3 chimaera). Dimeric and pentameric type I constructs are novel structures. We found that, similarly to full-length TSP1, intact trimeric type I repeats were inhibitory to HMVEC angiogenesis in vitro. However, dimeric and pentameric type I repeats of TSP1 only partially inhibited HMVEC proliferation and sprouting in vitro. TSP3, which is lacking type I repeats, had no inhibitory activity, confirming that type I repeats elicit the anti-angiogenic activity of TSP1.