Metastasis is a highly complex process involving the survival of tumor cells, both in the blood stream and within specific organs. Cell-death and survival are determined by a number of gene products from an expanding family of the Bcl-2 gene, either promoting or preventing apoptosis. Furthermore, the survival of tumor cells may favor the accumulation of additional genetic alterations causing further growth and invasive opportunities which may lead to metastasis. To examine whether the prevention of cell-death influences the metastatic behavior, we transfected a human breast cancer cell line MDA-MB-435 with the Bcl-x L cDNA and then studied metastatic ability of the selected clones in vivo. Our results show that Bcl-x L -clones had a decreased tumor growth latency and an increased metastatic ability. Apoptosisresistance to cytokines was induced in 435 cells by Bcl-x Lexpression with minor modifications in their proliferation rates. These cells also showed diminished adhesion to extracellular matrix proteins and a survival advantage in suspension over 435/Neo cells. Moreover, to determine survival in blood stream and in cells lodged in the lungs, we injected 435/Bcl-x L and 435/Neo cells at 1 : 3 proportion i.v., and animals were killed at intervals of 15' to 16 h after injection. Tumor cells were recovered from the lungs and Southern-blot analysis revealed the presence of exogenous Bcl-x L cDNA. These results showed that 435/Bcl-x L cells had a survival advantage in circulation over 435/Neo cells. This advantage in vivo was attributable to Bcl-x L expression. We conclude that Bcl-x L expression in breast cancer cells can increase metastatic activity. This advantage could be created by inducing resistance to apoptosis against cytokines, increasing cell survival in circulation, and enhancing anchorageindependent growth.Cell Death and Differentiation (2000) 7, 350 ± 359.

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Lysinuric protein intolerance (LPI) is a rare autosomal inherited disease caused by defective cationic aminoacid transport 4F2hc/y þ LAT-1 at the basolateral membrane of epithelial cells in the intestine and kidney. LPI is a multisystemic disease with a variety of clinical symptoms such as hepatosplenomegaly, osteoporosis, hypotonia, developmental delay, pulmonary insufficiency or end-stage renal disease. The SLC7A7 gene, which encodes the y þ LAT-1 protein, is mutated in LPI patients. Mutation analysis of the promoter localized in intron 1 and all exons of the SLC7A7 gene was performed in 11 patients from 9 unrelated LPI families. Point mutation screening was performed by exon direct sequencing and a new multiplex ligation probe amplification (MLPA) assay was set up for large rearrangement analysis. Eleven SLC7A7-specific mutations were identified, seven of them were novel: p.L124P, p.C425R, p.R468X, p.Y274fsX21, c.625 þ 1G4C, DelE4-E11 and DelE6-E11. The novel large deletions originated by the recombination of Alu repeats at introns 3 and 5, respectively, with the same AluY sequence localized at the SLC7A7 3 0 region. The novel MLPA assay is robust and valuable for LPI molecular diagnosis. Our results suggest that genomic rearrangements of SLC7A7 play a more important role in LPI than has been reported, increasing the detection rate from 5.1 to 21.4%. Moreover, the 3 0 region AluY repeat could be a recombination hot spot as it is involved in 38% of all SLC7A7 rearranged chromosomes described so far.

Cystinuria is a hereditary disorder caused by a defect in the apical membrane transport system for cystine and dibasic amino acids in renal proximal tubules and intestine, resulting in recurrent urolithiasis. Mutations in SLC3A1 and SLC7A9 genes, that codify for rBAT/b 0,ϩ AT transporter subunits, cause type A and B cystinuria, respectively. In humans, cystinuria treatment is based on the prevention of calculi formation and its dissolution or breakage. Persistent calculi are treated with thiols [i.e., D-penicillamine (DP) and mercaptopropionylglycine (MPG)] for cystine solubilization. We have developed a new protocol with DP to validate our Slc7a9 knockout mouse model for the study of the therapeutic effect of drugs in the treatment of cystine lithiasis. We performed a 5-wk treatment of individually caged lithiasic mutant mice with a previously tested DP dose. To appraise the evolution of lithiasis throughout the treatment a noninvasive indirect method of calculi quantification was developed: calculi mass was quantified by densitometry of X-ray images from cystinuric mice before and after treatment. Urine was collected in metabolic cage experiments to quantify amino acids in DP-treated and nontreated, nonlithiasic mutant mice. We found significant differences between DP-treated and nontreated knockout mice in calculi size and in urinary cystine excretion. Histopathological analysis showed that globally nontreated mutant mice had more severe and diffuse urinary system damage than DP-treated mice. Our results validate the use of this mouse model for testing the efficacy of potential new drugs against cystinuria.

Metastatic progression in ductal breast carcinomas are related to apoptosis in primary tumors. Frameshift mutations in a single-repeat sequence within the coding region (G)8 of the pro-apoptotic Bax gene have been related to microsatellite instability (MSI) and progression of some carcinomas and lymphomas. The aim of this study was to explore whether the extended lifespan of breast cancer cells can also be triggered by Bax mutation in ductal-breast carcinomas, and whether breast cancer cell MSI is related to the loss of apoptosis. For this purpose we studied frameshift mutations of a microsatellite (G)8 in the third exon of the Bax gene in a series of 105 ductal breast carcinomas, at T1 and T2-3 stages, 45 of which had lymph node metastasis. We analyzed MSI in five sequences of DNA isolated from normal and tumor tissue samples taken from 86 patients, and we explored the relationship between MSI and tumor apoptosis status. Bax mutation was not present in ductal breast carcinomas. MSI (two or more markers altered) was detected in 11.6% of tumors. Loss of apoptosis occurred in 80% (8/10) tumors with MSI, versus 17.8% of tumors without MSI (chi2 test, p = 0.0004), independently of Bax protein expression. We conclude that frameshift mutations of a microsatellite (G)8 of the Bax gene are not critical for the loss of apoptosis in breast cancer, and that loss of apoptosis may be a consequence of overexpression of anti-apoptotic protein Bcl-2 or Bcl-xL. Moreover, MSI in breast carcinomas might be the cause of loss of an apoptotic pathway that is not induced by frameshift mutations of a microsatellite (G)8 of the Bax gene.