Variability of PDYN and OPRK1 genes in four argentinian populations and its genetic association with clinical variables related to acute postsurgical pain / Variabilidad de los genes PDYN y OPRK1 en cuatro poblaciones argentinas y su asociación con variables clínicas relacionadas al dolor agudo post-quirúrgico

ABSTRACT Several population studies showed an association between variation in pain sensitivity and genetic polymorphisms located in Prodynorphin (PDYN) and Kappa Opioid Receptor (OPRK1) human genes. We analysed polymorphisms of these two genes to characterise their variation in Argentinian populations, as well as to evaluate their association with acute pain sensitivity. We studied 11 genetic markers in individuals from four locations in Argentina (Ciudad Autónoma de Buenos Aires, La Plata, Resistencia, and Misión Nueva Pompeya), calculated the population parameters, and evaluated the possible association among pain sensitivity, clinical, and genetic variables through a Generalised Estimating Equation model. High linkage disequilibrium was observed in the four populations for both genes, and significant differences were found among frequencies of Argentinian populations and those from other continents reported in the 1000 Genomes Project. Four PDYN gene polymorphisms from 3´ untranslated region and exon 4 showed association with acute pain sensitivity. One genotype of each of these polymorphisms was associated with a higher pain sensitivity, probably related with the activation of the N-methyl-D-aspartate (NMDA) receptors. We found a strong association with acute pain for the following clinical variables: 1) time after surgery, 2) intravenous klosidol supplied every 8 h, and 3) type of incision. Our results highlight the importance of a regional study of genetic variants which influence pain sensitivity and analgesic response.

RESUMEN La asociación entre la sensibilidad al dolor y los polimorfismos que presentan los genes humanos de prodinorfina (PDYN) y receptor opioide kappa (OPRK1) se ha evidenciado en distintos estudios poblacionales. Con el objetivo de caracterizar la variación de estos genes y evaluar su asociación con dolor agudo en la población argentina, analizamos 11 polimorfismos en individuos provenientes de cuatro localidades argentinas (Ciudad Autónoma de Buenos Aires, La Plata, Resistencia, y Misión Nueva Pompeya). Calculamos los parámetros poblacionales y evaluamos la posible asociación entre sensibilidad al dolor, variables clínicas y variables genéticas a través de un modelo de ecuación generalizada de estimación. Se observó alto desequilibrio de ligamiento para ambos genes en las cuatro poblaciones analizadas, y se encontraron diferencias significativas entre las frecuencias de poblaciones argentinas y las reportadas en el Proyecto 1000 Genomes para poblaciones de otros continentes. Cuatro polimorfismos de la región 3´UTR y el exón 4 de PDYN mostraron asociación con la sensibilidad al dolor agudo. En cada uno de estos polimorfismos, un genotipo resultó asociado con alta sensibilidad al dolor, probablemente en relación con la activación de receptores N-metil-D-aspartato (NMDA). Encontramos una fuerte asociación con dolor agudo para las siguientes variables clínicas: 1) tiempo post-cirugía, 2) administración intravenosa de klosidol cada 8 h, y 3) tipo de incisión. Nuestros resultados resaltan la importancia de realizar estudios regionales de variables genéticas que influyen en la sensibilidad al dolor y la respuesta analgésica.

Reactivity of monoclonal antibody FC-2.15 against drug resistant breast cancer cells. Additive cytotoxicity of adriamycin and taxol with FC-2.15.

Monoclonal antibody (MAb) FC-2.15 recognizes Lewis x antigen (Le(x)-Ag) expressed on the cell surface of most human breast cancer cells. FC-2.15 displays important human complement (C')-mediated cytotoxicity (CMC) against its target cells. In this study the reactivity of FC-2.15 against drug resistant-breast cancer cells was investigated, as well as the possibility to combine the antitumor activities of this MAb with adriamycin (Adr) or taxol. Since resistant clones with altered expression of tumor-associated antigens usually emerge after chemotherapy, the expression of Le(x)-Ag was analyzed in Adr(R) MCF-7 breast cancer cells (Adr resistant subline) and in tumor samples from nine patients with locally advanced breast carcinoma who were treated with FEC chemotherapy. A flow cytometry assay showed that most of Adr(R) MCF-7 cells, as well as wild type (WT) cells, expressed Le(x)-Ag; however, the Le(x) epitope is probably bound to different backbones in these cells. When the cytotoxic ability of FC-2.15 against WT and Adr(R) MCF-7 cells was compared, it was found that a 90 min treatment with FC-2.15 plus C' induced similar CMC against both cell lines. An important cytolysis was obtained at 5 microg/ml FC-2.15, reaching a plateau at 25 microg/ml, at which cell population was diminished to 21.1% for WT and 27.9 for Adr(R) MCF-7 cells. Regarding human tumors, Le(x)-Ag expression was evaluated in samples obtained before and in most cases after chemotherapy, and it could be observed that: 1) before treatment, tumor samples from all patients analyzed (responders and non-responders to chemotherapy) were FC-2.15-positive; 2) the presence of Le(x)-Ag was not modified after treatment. The combined action of Adr or taxol with FC-2.15 was then evaluated. WT and Adr(R) MCF-7 cells were cultured with Adr or taxol followed by an incubation with different FC-2.15 concentrations plus C'. When the effect of Adr alone was determined, ID50 were 1 x 10(-7) M for WT and 4.2 x 10(-5) M for Adr(R) MCF-7 cells. The cytotoxic ability of taxol alone was also tested, and ID50 were 6.4 x 10(-9) M for WT and 3.1 x 10(-6) M for Adr(R) MCF-7 cells. When FC-2.15 was added to Adr or taxol, the cytotoxicity of the drug-FC-2.15 combined treatment was always higher than the isolated effects, showing additive cytotoxicity at the different concentrations tested and with both cell lines. Our results suggest that FC-2.15 may be a useful agent against breast tumor cells which survive chemotherapy with Adr or taxol.

Description of a new human breast cancer cell line, IIB-BR-G, established from a primary undifferentiated tumor.

The establishment of a new human breast cancer cell line (IIB-BR-G) was successful after a previous growth of the cells isolated from a breast primary tumor in a female nude mouse. The IIB-BR-G cell line and the primary tumor do not express estrogen or progesterone receptors. Vimentin and keratin expression were found in the cell line and in the nude mouse tumor. This cell line displays high morphological heterogeneity with atypical multinucleated megacells, and it is capable of anchorage-independent growth and tumor formation in nude mice. The cytogenetic analysis confirmed its human origin and revealed multiple marker chromosomes and extensive chromosomal alterations including rearrangements, gains, losses, isochromosomes, and double minutes (DMs).

The expression of progesterone receptors coincides with an arrest of DNA synthesis in human breast cancer.

Two main models to account for the heterogeneous expression of estrogen receptors (ER) and progesterone receptors (PR) in human breast cancer have been proposed: the clonal model and the stem cell model. The authors previously provided evidence supporting the stem cell model since it was found that most of the proliferating cells in ER-positive (ER+) human breast cancer lack ER and that the ER-negative (ER-) and ER+ subpopulations are interrelated. The authors have analyzed in eighteen ER+/PR+ primary breast tumors the simultaneous expression of ER or PR (by immunohistochemistry) and DNA synthesis (by autoradiography) after 30 minutes of 3H-thymidine incorporation. The authors demonstrated that: (1) the average numbers of ER+ and PR+ cells were similar (36.8 +/- 10.7% and 39.3 +/- 17.6%, respectively); (2) The thymidine-labeling indexes of the ER+, ER-, PR+, and PR- subpopulations were 0.53 +/- 0.69%, 0.74 +/- 0.49%, 0.21 +/- 0.21 and 0.94 +/- 0.54%, respectively; and (3) 75.2% of the DNA-synthesizing cells were ER-, and 88.8% of them were PR-. The authors conclude that the cellular subpopulations expressing ER and PR were not identical, and the expression of PR was associated with a lower rate of cellular proliferation than was ER expression.