Jovita 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.

Organization of the histone genes in the rainbow trout (Salmo gairdnerii)

SummaryTwelve clones containing histone genes were isolated from a genomic trout library constructed in the vector Charon 4A. Each of the clones was found to contain a conserved 10.2-kb Eco RI fragment that contained one copy of each of the histones in the order H4-H2B-H1-H2A-H3, all of which are transcribed from the same strand. Genomic Southern blots indicate that these clusters are representative of the vast majority of the histone genes in the trout. Tandemly linked clusters were not found. Approximately 145 copies of this cluster are present in a trout sperm cell. Sequence analysis has shown the genes to be without introns and to show strong selection for codons ending in C or G. Consensus signals similar to those found in other histone genes are present in the flanking regions.

An H1 histone gene from rainbow trout (Salmo gairdnerii)

SummaryA 1.7-kbp DNA region from the 10.2-kb cluster containing the five rainbow trout histone genes has been subcloned in pBR322 and completely sequenced. It contains a trout histone H1 gene together with its 5′ and 3′ flanking sequences. This H1 gene codes for a H1 variant different from the major trout testis H1 previously sequenced by Macleod et al. (1977). Northern blots of total RNA from trout testis, kidney, and liver indicate that this H1 gene is expressed in all three tissues but that the level of H1 mRNA is much higher in testis than in other tissues. The lack of heterogeneity in the sizes and 5′ initiation sites of trout H1 mRNAs is surprising in view of the substantial heterogeneity of H1 variant proteins observed previously. The coding sequence of the H1 gene shows strong evidence of repeated partial duplications of a hexapeptide motif of the form Ala.Ala.Ala.Lys.Lys.Pro and of a pentapeptide phosphorylation-site sequence, Lys.Ser.Pro.Lys.Lys, during its evolution. Comparisons are drawn between this gene and the coding sequences of other vertebrate H1 genes from chicken andXenopus, and a strong homology is seen in the region of amino acids 22–101, which form the hydrophobic “head” of the H1 molecule. The 5′ and 3′ regulatory signals in the trout H1 are also compared with those of H1 genes from other sequences.

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.

Phylogeny and ontogeny of the phosphoglycerate mutases—IV. Distribution of glycerate-2,3-P2 dependent and independent phosphoglycerate mutases in algae, fungi, plants and animals

1. The distribution of the glycerate-2,3-P2 dependent and independent phosphoglycerate mutases (PGM) has been studied in more than eighty species. 2. PGM activity in the extracts has been measured in the presence and in the absence of glycerate-2,3-P2, at pH 7.5 and at pH 8.5. 3. All samples with glycerate-2,3-P2 dependent PGM possess higher activity at pH 7.5 than at pH 8.5. In contrast, samples with glycerate-2,3-P2 independent PGM possess lower activity at pH 7.5 than at pH 8.5. 4. In algae and fungi both glycerate-2,3-P2 dependent and independent PGM have been found. 5. In plants only glycerate-2,3-P2 independent PGM has been detected. 6. In animals both types of PGM are present. Independent PGM activity is present in sponges, coelenterates, myriapods, arachnids and echinoderms. Glycerate-2,3-P2 dependent PGM is present in platyhelminths, mollusks, annelids, crustaceans, insects and vertebrates.

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.

Organization and nucleotide sequence of rainbow trout histone H2A and H3 genes

SummaryA 2.56-kbp fragment containing genes coding for histones H2A and H3 that forms a portion of the 10.2-kbp cluster containing all five histone genes isolated from a λ-Charon 4A library of rainbow trout genomic DNA has been characterized in detail and its complete nucleotide sequence determined. The genes are arranged in tandem, being encoded on the same DNA strand. They are separated by 380 bp of intergenic spacer DNA that contains an alternating purine-pyrimidine stretch of 20 bp and a 46-bp stretch that has the potential of forming a triple cruciform structure. The histone genes contain no introns, have the RNA polymerase II promoter-associated signals known as CAAT and TATA boxes in their 5′ flanking regions and contain a conserved inverted repeat sequence, similar to that found in histone genes of other species, capable of forming a hairpin structure at the 3′ end of the transcription unit.

Phylogeny and ontogeny of the phosphoglycerate mutases—II. Characterization of phosphoglycerate mutase isozymes from vertebrates by their thermal lability and sensitivity to the sulfhydryl group reagents

Abstract 1. 1. To characterize the three phosphoglycerate mutase (PGM) isozymes present in vertebrates (types M, B and MB) their sensitivity to the reagents of the sulfhydryl groups and to heat treatment has been studied. 2. 2. In mammals and reptiles type M PGM is not affected by the —SH group reagents, type MB PGM is inhibited about 50% and type B PGM is fully inhibited. Types B and MB PGM show greater heat lability than type M PGM. 3. 3. In amphibians and fishes PGM isozymes do not differ in their sensitivity to the —SH reagents. 4. 4. The results strongly support the homodimeric and heterodimeric structure suggested for PGM isozymes and favour their genetic origin.