Proceso común

Following the isolation o f the RET oncogene Takahashi and colleagues demonstrated by northem blot analysis the presence o f multiple forms o f c-RET mRNA in the SK- N-SH Neuroblastoma, H t^60 Promyelocytic leukaemia, THP-1 Monocytic Leukaemia lines and mouse spinal cord (Takahashi Cooper 1987; Takahashi et al., 1988). No signal was detected from RNA isolated from 25 other cell lines and various other mouse tissues examined. Four transcripts o f c-RET mRNA o f 7.0, 6.0, 4.5 and 3.9 kb are present in normal tissues (Pachinis et al., 1993), primary tumours (Santoro et al., 1990; Nagao et al., 1992; Jhiang et al., 1992; Lanzi et al., 1992; Borrello et al., 1993; Matias-Guiu et al., 1995) and in many neuroblastoma (Ikeda et al., 1990; Nakamura et al., 1994), pheochromocytoma (Nakamura et al., 1994) and medullary thyroid carcinoma lines (Santoro et al., 1993a; Nakamura et al., 1994).

cDNA cloning and sequence analysis established the different c-RET mRNAs were derived from a primary transcript by differential splicing and polyadenylation (Tahira et al., 1990). This analysis suggest the c-ret locus to encode at least two (Tahira et al., 1990) and a possible third (Myers et al., 1995) intracellular, COOH-terminal isoforms and three other extracellular domain isoforms (Lorenzo et al., 1995; Xing et al., 1994). Tahira et al. (1990) first described two carboxyl-terminus splice variants o f the intracellular domain which differed by 51 amino acids. Nine o f the carboxyl-terminus amino acids o f the short isoform is replaced by a fifty-one amino acid segment which contains two additional tyrosine residues in the long isoform m RNA By Northem blot analysis using different fragments o f cDNAs as probes, Tahira and colleagues suggests the 4.5 kb, 3.9 kb and possibly the 7.5 kb transcripts to encode the short isoform o f

1072 amino acid residues, and the 6.0 kb and 4.5 kb transcripts to encode the long isoform o f 1114 residues. Recently Myers et al. (1995) reported the presence o f a novel exon 19 splice variant, containing a unique 41 amino acid carboxyl-terminus, in pheochromocytoma and neuroblastoma lines.

In addition to the COOH-terminal isoforms, splice variations predicted to encode extracellular ligand binding domain isoforms has also been reported (Lorenzo et al., 1995; ]üng et al., 1994). These transcripts, two predicated to encode truncated membrane spanning proteins lacking the extracellular ligand-binding domain, and a third predicated to encode a soluble secreted form of the receptor, has been shown to be present in normal and tumour tissues (Lorenzo et al., 1995). Xing et al. (1994) reported alternative splicing involving introne 4 sequences to generated extracellular domain variants possing either a 62 bp or 69 bp insertion. The presence of these heterogeneous populations o f c-RET mRNA imply that, like the multiple isoforms of Trk proteins generated by differential alternative splicing (reviewed by Hanks, 1991), these splice variants may represent stable translatable messages which encode for distinct functional proteins.

E Protein Encoded by the c-RET Proto-Oncogene

Western blot analysis using intracellular domain anti-RET antibodies detects two phosphorylated glycosylated proteins of 170 kDa and 150 kDa in human (Takahashi et al., 1991; Miyazaki et al., 1996), and 160 kDa and 140 kDa in mouse (Iwamoto et al., 1993a), neuroblastoma lines. However, in lysate of a human neuroblastoma cell line grown in the presence of tunicamycin, an inhibitor of N-linked glycosylation, a protein of 120 kDa was detected. This is the size expected from the deduced amino acid sequences of the gene. Therefore the two isoforms of the c-RET protein detected using intracellular domain anti-RET antibodies represent differential glycosylation products. Consistent with this twelve possible N-linked glycosylation sites have been identified within the amino acid sequence of murine c-RET protein (Iwatoma et al., 1993a). Given their similar sizes, both the long and short isoforms are represented by the 120 kDa non-glycosylated protein band.

E The Hunum c-RET Locus

After isolating the ret II oncogene, Ishizaka and colleges cloned the c-RET proto­ oncogene and mapped the position of the human c-RET locus to chromosome lO.ql 1

Fig 1.3 Genomic structure o f the human c-RET proto-oncogene.

Restriction enzyme map of a 150kb region of the human genome containing the C- RET proto-oncogene.

EcoRl (E), N o tl M ini and A W restriction sites. ATG: translation initiation site. CEN: Centermeric end. TEL: Telemeric end.

The C-RET proto-oncogene comprise of twenty exons (black squares) with a CA repeat in introne 5 and two other CA repeats flanking the gene.

m u HI

10 4.8 4.2

1.2 3.90.8 I5.8

(CA)n I n tr o n H :

RET-1 NTS site of R E T

rea rra n gem en ts in PTC

(CA)n S-TCL2

near the MENE locus by fluorescence in situ hybridisation (FISH) (Ishizaka et al., 1989; Sozzi et al., 1991). This region of the human genome, where the MENII locus had previously been mapped by linkage analysis (Masthew et. al., 1987; Simson et al.,

1987), has been sub-cloned as a contig of yeast artificial chromosomes (YACs) containing cytogenetic markers linked with MEN2A and c-RET (Lairmore et al., 1993; Brooks-Wilson et al., 1993; Mole et al., 1993). Around this time Itoh et al. (1992) characterised the human c-RET promoter to lack the TATA initiation m otif with high GC content. Ceccherini et al. (1993) using a PCR and sequencing approach and Kwok et al. (1993) employing an exon trapping methodology reported the structure o f the human c-RET locus to consist of 20 exons distributed along 30 kb of genomic DNA.

Recently the complete genomic structure of the c-RET locus has been compiled by Pasini et al.(1995). These workers constructed cosmid mini-libraries from the YAC contig around the MENE locus (Mole et al., 1993) and compiled a cosmid contig spanning 150 kb of the genome containing the c-RET proto-oncogene. Fig 1.4 illustrates the structure of the human c-RET locus. The 20 exons of the gene (Kwok et al., 1993) are distributed along 60 kb of the genome (Pasini, 1995). Introne 1 accounts for 24 kb of the locus and the remainder of the gene is contained within 31 kb o f the locus. The overall genomic structure, with large first introne and more clustered 3' exons, is reminiscent of PDGFRB (Sherr, 1990) and c-Kit (Andre et al, 1992; Vandenbark et al, 1992) which also encode receptor tyrosine kinases (Pasini, 1995).