Tomoko Nakata

Fusion of a novel gene, ELKS, to RET due to translocation t(10;12)(q11;p13) in a papillary thyroid carcinoma.

In papillary thyroid carcinomas, the genes for receptor‐type tyrosine kinase, RET or TRKA, are sometimes rearranged, resulting in fusion of its tyrosine kinase domain to 5′ portions of several activating genes. In a papillary thyroid carcinoma, we identified a novel gene (ELKS), the 5′ portion of which is fused to the RET gene by gene rearrangement due to the translocation t(10;12)(q11;p13). Subsequent cloning of the ELKS cDNA revealed that ELKS encodes a novel 948 amino acid peptide and is expressed ubiquitously in human tissues. The presence of multiple coiled‐coil domains in the ELKS product suggests that the ELKS protein forms dimers. Since the tyrosine kinase of RET is activated by dimerization that occurs when its ligands bind to the receptor, fusion of RET with the 5′ dimerization domains of ELKS would activate its cytoplasmic tyrosine kinase constitutively in papillary thyroid carcinomas. Genes Chromosomes Cancer 25:97–103, 1999.

Cellular localization of prolactin-releasing peptide messenger RNA in the rat brain.

Prolactin-releasing peptide (PrRP), a novel peptide identified as the endogenous ligand for an orphan receptor isolated from the pituitary, is a potent stimulator of prolactin release. To get a clue of the functional roles of the peptide, we performed in situ hybridization histochemistry for PrRP mRNA to define the cellular localization of PrRP-producing cells in the brain of the cycling adult female rat during diestrus. The PrRP mRNA-containing cells were located in the caudal part of the dorsomedial nucleus of the hypothalamus. In the brainstem, the cells were found in the caudal part of the solitary tract nucleus and in the caudal ventrolateral medulla (ventrolateral intermediate reticular field). Specific signals for PrRP mRNA were not detected in other brain regions. Although PrRP is a candidate for being a hypophysiotropic specific releasing factor, the discrete distribution of PrRP in the extrahypothalamic area suggests that the peptide has other physiological functions in the central nervous system.

Prolactin secretion in response to prolactin-releasing peptide and the expression of the prolactin-releasing peptide gene in the medulla oblongata are estrogen dependent in rats

Prolactin-releasing peptide (PrRP), recently isolated from bovine hypothalamus as an endogenous ligand to a seven transmembrane-domain orphan receptor, is a candidate specific prolactin-releasing factor. The prolactin-releasing activity of the peptide and the expression of the PrRP gene were examined in vivo in relation to estrogen status. Plasma prolactin levels increased significantly with a peak at 5 min after the administration of 50 microg/kg PrRP in female rats in estrus under urethane anesthesia as compared with those in vehicle-treated control rats, but not in female rats in diestrus or proestrus or in male rats. In ovariectomized rats treated with supraphysiological concentration of estrogen, a dose-dependent increase of prolactin secretion in response to 2-50 microg/kg PrRP was observed. However, the peak values induced by 50 microg/kg PrRP were much less than those induced by 2 microg/kg thyrotropin-releasing hormone (TRH). PrRP mRNA levels in the medulla oblongata were decreased by ovariectomy and increased by estrogen treatment. The data indicate that estrogen is prerequisite to the stimulatory effect of PrRP on the secretion of prolactin and to the increase of PrRP mRNA levels in the medulla oblongata. The weak in vivo potency of PrRP on prolactin secretion relative to TRH suggests that PrRP differs from the classical hypophysiotropic hypothalamic releasing hormones.

Correlation of allelic losses and clinicopathological factors in 504 primary breast cancers.

Human breast cancers frequently show allelic loss or loss of heterozygosity (LOH) at specific chromosomal regions. To understand the possible role of these genetic alterations in tumor development and progression, we examined LOH at loci on chromosomal arms lp, 3p, 11p, 13q, 16q, 18q, and 22q in 140 to 246 cases of primary breast cancers and compared it with lymph node metastasis, histological type, tumor stage, estrogen receptor (ER) and progesterone receptor (PgR) status. LOH at 1p22-31 correlated with lymph node metastasis and a tumor size of greater than 2 cm. LOH at 13ql2-14 and 18q21 were most frequently observed in tumors of the solid-tubular type. LOH at 1p34-36 was more frequent in tumors of the scirrhous and solidtubular types than in other less aggressive histological types. Furthermore, a significant association was observed between LOH at 3pl4-21, 11p11-15 and 13ql2-14 and the absence of progesterone receptors. These results suggest that some clinical characteristics of breast cancers are determined by loss of tumor suppressor genes present at specific chromosomal regions.

Ret/PTC3 is the most frequent form of gene rearrangement in papillary thyroid carcinomas in Japan.

AbstractRearrangements of the RET and TRK proto-oncogenes, which generate fusion oncogenes, are frequent in papillary thyroid carcinomas in Caucasian populations. To determine the spectrum of gene rearrangements in Japanese patients, we systematically examined 40 papillary thyroid carcinomas for all possible types of gene fusion events involving RET or TRK genes. RET rearrangements were found in ten tumors (25%): ret/PTC1 had occurred in two tumors, ret/PTC2 in one, ret/PTC3 in six, and a novel RET rearrangement in the remaining patient. In this last patient, the 5′ novel sequence was fused in-frame to the RET amino acid sequence; thus, the fusion gene may encode a protein with a RET kinase domain at the carboxy terminus. The RET gene was fused to 5′ donor sequences at the beginning of exon 12 in all ten tumors. No rearrangements involving the TRK gene were found in this panel of carcinomas. Our results indicated that constitutive activation of the RET by gene rearrangement is a frequent mechanism of papillary thyroid carcinogenesis in Japanese adults.

Differential expression of multiple isoforms of the ELKS mRNAs involved in a papillary thyroid carcinoma

A novel gene, ELKS, whose 5′ portion was fused to the RET gene, was found in a papillary thyroid carcinoma. A cDNA of this gene obtained from a human‐brain cDNA library revealed that it encoded a peptide of 948 amino acids, termed ELKSα. We identified four other isoforms, which encoded ELKSβ, ELKSγ, ELKSδ, and ELKSϵ proteins consisting, respectively, of 992, 720, 1088, and 1116 amino acid residues. Analysis of the gene structure revealed that the isoforms were generated by alternative splicing. Isoforms β, γ, δ, and ϵ all contain an optional exon (exon14a), but ELKSγ, ‐δ, and ‐ϵ lack exon 1b. ELKSγ lacks exons 3 to 6. ELKSδ and ‐ϵ lack exons 12 and 17; ELKSϵ contains an optional exon (exon 6a). Analysis by RT‐PCR suggested that ELKSα and ELKSβ mRNAs are abundant in the brain, ELKSδ and ELKSϵ mRNAs predominate in testis and thyroid, and ELKSϵ mRNA predominates in other tissues. To prove whether the fusion of different ELKS isoforms to RET (between ELKS coiled‐coil domains and the RET kinase domain) could produce chimeric proteins that could be autophosphorylated, we synthesized ELKSγ‐RET, ELKSδ‐RET, and ELKSϵ‐RET fusion proteins in vitro. Immunoblotting with anti‐ELKS, anti‐RET, and anti‐phosphotyrosine antibodies demonstrated that the chimeric proteins were constitutively phosphorylated at tyrosine residues, whereas native RET protein was not. These results indicate that the ELKS gene is alternatively spliced, and that every type of ELKS‐RET chimeric protein having oligomerization domains can activate RETs cytoplasmic tyrosine kinase.

Multiplex Mutation Screening of the BRCA1 Gene in 1000 Japanese Breast Cancers

To detect BRCA1 mutations in Japanese breast cancer patients, we screened 1,000 unselected primary cancers for mutations in exon 11, which accounts for 61% of the entire BRCA1 coding sequence. Using a method based on multiplex single‐strand conformational polymorphism (SSCP) analysis of multiple restriction fragments generated by restriction‐enzyme digestion of amplified DNA, we identified eight mutations. All eight were germline mutations; four of them were non‐sense mutations or small deletions resulting in premature stop codons, and the other four were missense mutations. The Japanese carriers of these mutant BRCA1 alleles had developed breast cancers at ages ranging from 45 to 62, five of them bilaterally.

Genomic organization and chromosomal mapping of ELKS, a gene rearranged in a papillary thyroid carcinoma

AbstractWe recently isolated a novel cDNA, designated ELKS, that was fused to RET cDNA in a papillary thyroid carcinoma. Its encoded polypeptide sequence was rich in glutamic acid (E), leucine (L), lysine (K), and serine (S), and was characterized by the presence of nine alpha-helical coiled-coil domains consisting of periodic heptad repeats. We have now cloned the entire structure of the human ELKS gene from within a 700-kb genomic region represented by overlapping bacteriophage P1-derived artificial chromosome (PAC) and bacterial artificial chromosome (BAC) clones, and localized it to chromosomal band 12p13.3 by fluorescence in situ hybridization. The gene is approximately 500 kb long, with 19 exons and 18 introns; the transcription initiation site within exon 1 is separate from the initiation codon (in exon 2). Analysis of the exon/intron structure revealed that introns interrupt the coding sequence in such a way that many functional segments of the protein are encoded by distinct exons. Exon 1 encodes the 5′ non-coding region; exons 2, 3, 6, 7, 8, 9, 11, 14, and 15 encode the nine coiled-coil domains. Exons 17–19 constitute the 3′ non-coding region. Analysis of the region immediately upstream of exon 1 showed that it was extremely rich in G/C nucleotides and contained multiple Sp-1 and AP2 binding sequences. The ELKS-RET gene fusion rearrangement we had observed in a papillary thyroid carcinoma occurred between intron 10 of the ELKS gene and intron 11 of RET.

Identification of a New Commonly Deleted Region within a 2-cM Interval of Chromosome 11p11 in Breast Cancers

Allelic loss has been observed on the short arm of chromosome 11 in a variety of human cancers. We have examined 184 breast cancers for allelic loss anywhere in chromosome 11p, using 15 well-spaced microsatellite markers. Allelic loss was observed in 86 cases (47%) and a new commonly deleted region 2-cM in length was identified at 11p11 between loci D11S986 and D11S1313, in addition to a 12-cM region of a common deletion at 11p15.5. A significant association was found between allelic loss on 11p15.5 and LOH on 11p11 and the loss of progesterone receptors.

Microinjection of dihydrotestosterone into the medial preoptic area produces male-like pattern of growth hormone secretion in ovariectomized female rats.

The pattern of growth hormone (GH) secretion is sexually dimorphic in rats. We have previously shown that the secretory pattern in adult ovariectomized (OVX) female rats is masculinized by the administration of a single dose of dihydrotestosterone (DHT), a nonaromatizable androgen. To investigate the primary site of action of DHT in the brain, a small amount of DHT was injected directly into a defined area of the brain, and the blood GH profile was observed for 18 h in conscious adult OVX female rats. The bilateral direct injection of 1 µg DHT into the medial preoptic area (MPA) produced a male-like secretory pattern of GH in OVX rats. The masculinizing effects became apparent at 9 h after injection, from which time the episodic GH secretion was produced regularly at intervals of about 150 min, the amplitude of the peak increased and baseline levels were lowered. These parameters, analyzed during 9–18 h after DHT injection, were not different from those in adult male rats. On the contrary, microinjection of DHT into the bed nucleus of the stria terminalis, the hypothalamic periventricular nucleus, or the hypothalamic arcuate-ventromedial nucleus did not affect the secretory pattern of GH. The data indicate that DHT primarily acts on cells in the MPA through androgen receptors and modulates the secretion of somatostatin and/or GH-releasing hormone secondarily to masculinize the GH secretory pattern in OVX rats.