Cristóbal Mezquita

Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT) and a truncated isoform of VEGFR-1 (i21-VEGFR-1), which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer.

Inhibition of the Canonical IKK/NFκB Pathway Sensitizes Human Cancer Cells to Doxorubicin

The NFκB family is composed by five subunits (p65/RelA, c-Rel, RelB, p105-p50/NFκB1, p100-p52/NF-κB2) and controls the expression of many genes that participate in cell cycle, apoptosis, and other key cellular processes. In a canonical pathway, NF-κB activation depends on the IKK complex activity, which is formed by three subunits (IKKα and IKKβ and IKKγ/NEMO). There is an alternative NFκB activation pathway that does not require IKKβ or IKKγ/NEMO, in which RelB is a major player. We report in a panel of human breast cancer cells that the IKK/NFκB system is generally overexpressed in breast cancer cells and there is heterogeneity in expression levels of individual members between different cell lines. Doxorubicin, an anticancer agent used in patients with breast cancer, activated NFκB and appeared to be less effective in cells expressing predominantly members of the canonical IKK/NFκB. Two NFκB inhibitors, bortezomib and NEMO-Binding Domain Inhibitory Peptide, prevented doxorubicin-induced NFκB activation and increased doxorubicin antitumor effects in BT-474 cells. Transient downregulation of members of the canonical pathway (p65, p52, c-Rel and IKKγ/NEMO) by siRNA in HeLa cells increased doxorubicin cytotoxicity. In contrast, silencing of RelB, a key subunit of the alternative pathway, had no evident effects on doxorubicin cytotoxicity. To conclude, NFκB inhibition sensitized cells to doxorubicin, implying directly p65, p52, c-Rel and IKKγ/NEMO subunits in chemoresistance, but not RelB. These findings suggest that selective inhibition of the canonical NFκB pathway is sufficient to improve doxorubicin antitumor effects.

SEVERAL NOVEL TRANSCRIPTS OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE EXPRESSED IN ADULT CHICKEN TESTIS

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), in addition to being a classic glycolytic enzyme, is a multifunctional protein involved in relevant cell functions such as DNA replication, DNA repair, translational control of gene expression, and apoptosis. Although the multifunctional nature of GAPDH suggests versatility in the mechanisms regulating its expression, no major qualitative changes and few quantitative changes in the GAPDH transcripts have been reported. While studying the expression of GAPDH during spermatogenesis, we detected alternative initiations to TATA box and alternative splicings in the 5′ region of the pre‐mRNA, resulting in at least six different types of mRNAs. The amount and the polyadenylation of the GAPDH transcripts increased in mature testis in relation to immature testis and further increased when cell suspensions from mature testis were exposed to heat shock. These results suggest that alternative initiation, alternative splicing, and polyadenylation could provide the necessary versatility to the regulation of the expression of this multifunctional protein during spermatogenesis. J. Cell. Biochem. 71:127–139, 1998.

Changes in nuclear content of protein conjugate histone H2A—ubiquitin during rooster spermatogenesis

Electrophoretic analysis of acid‐soluble chromosomal proteins isolated from rooster testis cell nuclei at different stages of spermatogenesis, revealed that the nuclear content of a protein identified by its solubility, electrophoretic mobility and amino acid analysis as the protein conjugate histone H2A—ubiquitin (uH2A, A24) changed markedly from meiotic cells to late spermatids. The protein was not detectable in tetraploid primary spermatocytes; it was present in 1.7% of the total amount of nucleosomal core histones in early spermatids and reached its maximum level (3.5% and 11%) at the end of spermiogenesis, when histones are replaced by the protamine galline.

A novel intracellular isoform of VEGFR-1 activates Src and promotes cell invasion in MDA-MB-231 breast cancer cells.

Two types of VEGFR‐1 receptors have been characterized: a full‐length transmembrane receptor and a truncated extracellular soluble isoform (sVEGFR‐1). We report here the characterization, in normal and cancer cells, of a new family of intracellular isoforms of VEGFR‐1 resulting from alternative initiation of transcription in intronic sequences of the gene. While the classical isoforms of VEGFR‐1 were barely detectable in MDA‐MB‐231 breast cancer cells, one of the intracellular isoforms transcribed from intron 21 (i21VEGFR‐1) was the main isoform expressed in these cells. The new transcript encodes for a protein that contains only the phosphotransferase domain and the carboxyterminal tail of VEGFR‐1. Treatment of MDA‐MB‐231 cells with siRNA specific for the tyrosine domain of VEGFR‐1 suppressed the expression of i21VEGFR‐1, downregulated phosphorylation of Src at tyrosine 418, and reduced markedly the invasion capacity of these cells in vitro. Accordingly, overexpression of transfected i21VEGFR‐1 in MDA‐MB‐231 cells upregulated the active form of Src and increased invasiveness of MDA‐MB‐231 cells. The expression of i21VEGFR‐1 in MDA‐MB‐231 cells was inhibited by retinoic acid. Both, activation of Src and downregulation by retinoic acid, have been reported in other intracellular members of the Fms/Kit/PDGFR family of tyrosine kinases, particularly in the intracellular isoform of c‐kit, analogous structurally to i21VEGFR‐1 and frequently expressed in cancer cells. J. Cell. Biochem. 110: 732–742, 2010.

Marked differences between avian and mammalian testicular cells in the heat shock induction and polyadenylation of Hsp70 and ubiquitin transcripts.

Mammalian male germ cells undergo apoptosis at the bodys internal temperature of 37°C. Birds, however, are unique among homeothermic animals in developing spermatogenesis at the elevated avian internal body temperature of 40–41°C. To shed light on the mechanisms that maintain an efficient avian spermatogenesis at elevated temperatures we compared, in mouse and chicken testicular cells, the expression of genes that are essential for stress resistance: Hsp70 and ubiquitin. While the expression of Hsp70 and ubiquitin did not change upon heat shock in mouse testicular cells, both the amount and polyadenylation of Hsp70 and ubiquitin transcripts increased when male germ cells from adult chicken testis were exposed to elevated temperatures.

Down‐regulation of Flt‐1 gene expression by the proteasome inhibitor MG262

The mechanisms involved in the anti‐angiogenic actions of the proteasome inhibitors are poorly understood. Here, we report that the gene expression of the VEGF receptor Flt‐1 (vascular endothelial growth factor receptor 1) was down‐regulated by the reversible proteasome inhibitor MG262 in explant cultures of the developing chicken pecten oculi, a vascular organ consisting of endothelial cells, pericytes, and macrophages. In addition, the inhibitor prevented the induction of Flt‐1 by lipopolysaccharide (LPS) in macrophages and down‐regulated the expression of Flt‐1 after LPS induction. Flt‐1 gene expression was also down regulated by MG262 in cultures of human microvascular endothelial cells. Interestingly, a transcript of Flt‐1, coding for a soluble form of the receptor (sFlt‐1) with anti‐angiogenic properties, was not down‐regulated in the same extent. Only a small decrease in the expression of VEGF and Ang‐2 was detected in the pecten oculi upon inhibition of the proteasome, while no major changes were observed in the expression of other angiogenic molecules, such as KDR or Ang‐1. Since recent experiments have demonstrated the importance of anti‐Flt‐1 therapy in the inhibition of tumor angiogenesis, retinal angiogenesis, arthritis, and atherosclerosis (Luttun et al. [ 2002 ]: Nat Med 8:831–840), our observation on down‐regulation of Flt‐1 in microvascular endothelial cells and macrophages by MG262 supports the postulated role of the proteasome inhibitors as potential candidates for therapeutic modulation of angiogenesis and inflammation.

Differential expression of Bcl-2 and Bcl-x during chicken spermatogenesis

We report the isolation and characterization of a chicken testis bcl‐xL cDNA coding for a long bcl‐x protein with a hydrophobic tail, and the expression of bcl‐2 and bcl‐x during chicken spermatogenesis. Bcl‐2 is highly expressed in embryonic and immature testes enriched in spermatogonia and barely detectable in mature testes, where most of the cells are meiotic and postmeiotic. Bcl‐x is expressed in both mature and immature testes, but in a lesser amount in mature testes. Differential expression of bcl‐2 and bcl‐x during spermatogenesis is consistent with the reported different susceptibility to apoptosis of spermatogonia, and meiotic and postmeiotic cells. Mol. Reprod. Dev. 47:26–29, 1997.

Four isoforms of the signal‐transduction and RNA‐binding protein QKI expressed during chicken spermatogenesis

Genes expressed during spermatogenesis undergo alternative initiation and alternative splicing and may be under the control of a coordinated mechanism of RNA processing. A family of proteins that combine features of signal‐transduction and RNA‐binding molecules could be instrumental in this process. We have characterized a cDNA from adult chicken testis that codifies a highly conserved member of the STAR protein family, the orthologue of the mouse quakinggene qkI. The predicted chicken protein differs only in four amino acids from the corresponding mouse protein. Messages of 7, 6, and 5 kb are expressed differentially during chicken spermatogenesis. The 5‐kb message, the predominant form in adult testis, presents heterogeneity in the coding region, showing insertions of 51 and 75 bp and a deletion of 24 bp, which gives rise to four possible isoforms of the protein. Mol. Reprod. Dev. 50:70–78, 1998.

Characterization of a chicken polyubiquitin gene preferentially expressed during spermatogenesis

We have previously reported that a chicken polyubiquitin gene (Ub II) not expressed under normal or heat shock conditions in chick fibroblasis is transcribed during spermatogenesis [(1987) Nucleic Acids Res. 15, 9604]. The level of Ub II mRNA is several‐fold higher in testis cells than in somatic tissues. The gene Ub II possesses characteristic features not seen in the polyubiquitin gene expressed in heat shock conditions (Ub I). The 5′ noncoding region of Ub II shows the consensus cAMP regulatory element (CRE) followed immediately downstream by a CA dinucleotide. It has been proposed that this extended CRE may be involved in the coordinate expression of various genes during spermatogenesis.