Functional analysis of the -2548G/A leptin gene polymorphism in breast cancer cells.

Leptin is overexpressed in human breast tumors and is produced by breast cancer cells in response to obesity-related stimuli. The leptin promoter polymorphism Lep-2548G/A can be associated with increased leptin secretion by adipocytes and elevated cancer risk. However, molecular mechanisms underlying the link between Lep-2548G/A and breast cancer have never been addressed. Lep-2548G/A is proximal to a binding site for the transcriptional factor Sp1. Furthermore nucleolin, a transcriptional repressor, can bind Sp1 or its consensus site. Consequently, we focused on the impact of Lep-2548G/A on Sp1- and nucleolin-dependent leptin transcription in breast cancer cells. The Lep-2548G/A was identified in a homozygous conformation in BT-474 and SK-BR-3 breast cancer cells, in a heterozygous conformation in MDA-MB-231 cells, and a wild-type Lep-2548G/G sequence was present in MCF-7 and ZR-75-1 cells. The occurrence of Lep-2548A/A and Lep-2548G/A coincided with high and intermediate leptin mRNA expression, respectively, while cells containing Lep-2548G/G expressed low leptin mRNA levels. We demonstrated that the existence of Lep-2548G/A improved efficient recruitment of Sp1 to DNA under insulin treatment, while Sp1 loading on DNA containing Lep-2548G/G was not insulin-dependent. In contrast, nucleolin binding to Lep-2548G/A was downregulated in response to insulin, while it was not regulated on Lep-2548G/G. The presence of Lep-2548G/A was studied in breast cancer epithelial cells by IHC and LCM. Interestingly, all 14 tumors expressing high leptin levels contained Lep-2548A/A. In conclusion, the occurrence of Lep-2548G/A can enhance leptin expression in breast cancer cells via Sp1- and nucleolin-dependent mechanisms and possibly contribute to intratumoral leptin overexpression.

Mechanism and tissue specificity of nicotine-mediated lung S-adenosylmethionine reduction.

We previously reported that chronic nicotine infusion blocks development of Pneumocystis pneumonia. This discovery developed from our work demonstrating the inability of this fungal pathogen to synthesize the critical metabolic intermediate S-adenosylmethionine and work by others showing nicotine to cause lung-specific reduction of S-adenosylmethionine in guinea pigs. We had found nicotine infusion to cause increased lung ornithine decarboxylase activity (rate-controlling enzyme of polyamine synthesis) and hypothesized that S-adenosylmethionine reduction is driven by up-regulated polyamine biosynthesis. Here we report a critical test of our hypothesis; inhibition of ornithine decarboxylase blocks the effect of nicotine on lung S-adenosylmethionine. Further support is provided by metabolite analyses showing nicotine to cause a strong diversion of S-adenosylmethionine toward polyamine synthesis and away from methylation reactions; these shifts are reversed by inhibition of ornithine decarboxylase. Because the nicotine effect on Pneumocystis is so striking, we considered the possibility of tissue specificity. Using laser capture microdissection, we collected samples of lung alveolar regions (site of infection) and respiratory epithelium for controls. We found nicotine to cause increased ornithine decarboxylase protein in alveolar regions but not airway epithelium; we conclude that tissue specificity likely contributes to the effect of nicotine on Pneumocystis pneumonia. Earlier we reported that the full effect of nicotine requires 3 weeks of treatment, and here we show recovery is symmetrical, also requiring 3 weeks after treatment cessation. Because this time frame is similar to pneumocyte turnover time, the shift in polyamine metabolism may occur as new pneumocytes are produced.

The T-cell receptor in primates: identifying and sequencing new owl monkey TRBV gene sub-groups.

The New World primate Aotus nancymaae (owl monkey) has been shown to be an excellent experimental model when studying malarial parasites. Characterising the T-cell receptor (TR) alphabeta repertoire by means of the different variable beta (TRBV) genes displayed contributes to a better understanding of these lymphocytes' role in the response against several malarial antigens. This study describes identifying and characterising eleven new TRBV gene sub-groups in cDNA from Aotus nancymaae's peripheral blood lymphocytes; these 11 gene sequences displayed homology to the previously reported human TRBV3, TRBV10, TRBV11, TRBV14, TRBV18, TRBV19, TRBV20, TRBV25, TRBV27, TRBV29 and TRBV30 sub-groups, resulting in 83% overall homology at the amino acid level. An additional Aotus sequence was found having similarity with the human TRBJ-2-7*01 gene. Evolutionary relationships amongst these sequences and the homologous genes from both New and Old World primates have shown that the TRBV repertoire has been maintained in the species being studied, displaying varying association patterns and substitution rates, depending on the sub-group being studied. The degree of identity observed when comparing human and Aotus genes suggests that these species might have a similar TRBV repertoire.