Kay Huebner

Cloning, Characterization, and Chromosomal Localization of a Human 5-HT6 Serotonin Receptor

Abstract: We describe the cloning and characterization of a human 5‐HT6 serotonin receptor. The open reading frame is interrupted by two introns in positions corresponding to the third cytoplasmic loop and the third extracellular loop. The human 5‐HT6 cDNA encodes a 440‐amino‐acid polypeptide whose sequence diverges significantly from that published for the rat 5‐HT6 receptor. Resequencing of the rat cDNA revealed a sequencing error producing a frame shift within the open reading frame. The human 5‐HT6 amino acid sequence is 89% similar to the corrected rat sequence. The recombinant human 5‐HT6 receptor is positively coupled to adenylyl cyclase and has pharmacological properties similar to the rat receptor with high affinity for several typical and atypical antipsychotics, including clozapine. The receptor is expressed in several human brain regions, most prominently in the caudate nucleus. The gene for the receptor maps to the human chromosome region 1p35–p36. This localization overlaps that established for the serotonin 5‐HT1Dα receptor, suggesting that these may be closely linked. Comparison of genomic and cDNA clones for the human 5‐HT6 receptor also reveals an RsaI restriction fragment length polymorphism within the coding region.

Meis1, a PBX1-related homeobox gene involved in myeloid leukemia in BXH-2 mice.

Leukemia results from the accumulation of multiple genetic alterations that disrupt the control mechanisms of normal growth and differentiation. The use of inbred mouse strains that develop leukemia has greatly facilitated the identification of genes that contribute to the neoplastic transformation of hematopoietic cells. BXH-2 mice develop myeloid leukemia as a result of the expression of an ecotropic murine leukemia virus that acts as an insertional mutagen to alter the expression of cellular proto-oncogenes. We report the isolation of a new locus, Meis1, that serves as a site of viral integration in 15% of the tumors arising in BXH-2 mice. Meis1 was mapped to a distinct location on proximal mouse chromosome 11, suggesting that it represents a novel locus. Analysis of somatic cell hybrids segregating human chromosomes allowed localization of MEIS1 to human chromosome 2p23-p12, in a region known to contain translocations found in human leukemias. Northern (RNA) blot analysis demonstrated that a Meis1 probe detected a 3.8-kb mRNA present in all BXH-2 tumors, whereas tumors containing integrations at the Meis1 locus expressed an additional truncated transcript. A Meis1 cDNA clone that encoded a novel member of the homeobox gene family was identified. The homeodomain of Meis1 is most closely related to those of the PBX/exd family of homeobox protein-encoding genes, suggesting that Meis1 functions in a similar fashion by cooperative binding to a distinct subset of HOX proteins. Collectively, these results indicate that altered expression of the homeobox gene Meis1 may be one of the events that lead to tumor formation in BXH-2 mice.

FHIT gene therapy prevents tumor development in Fhit-deficient mice

The tumor suppressor gene FHIT spans a common fragile site and is highly susceptible to environmental carcinogens. FHIT inactivation and loss of expression is found in a large fraction of premaligant and malignant lesions. In this study, we were able to inhibit tumor development by oral gene transfer, using adenoviral or adenoassociated viral vectors expressing the human FHIT gene, in heterozygous Fhit+/− knockout mice, that are prone to tumor development after carcinogen exposure. We therefore suggest that FHIT gene therapy could be a novel clinical approach not only in treatment of early stages of cancer, but also in prevention of human cancer.

WWOX gene restoration prevents lung cancer growth in vitro and in vivo

The WWOX (WW domain containing oxidoreductase) gene at the common fragile site, FRA16D, is altered in many types of cancer, including lung cancer. We have examined the tumor suppressor function of WWOX in preclinical lung cancer models. The WWOX gene was expressed in lung cancer cell lines through recombinant adenovirus (Ad) infection (Ad-WWOX), and through a drug [ponasterone A, (ponA)]-inducible system. After WWOX restoration in vitro, endogenous Wwox protein-negative cell lines (A549, H460, and H1299) underwent apoptosis through activation of the intrinsic apoptotic caspase cascade in A549 and H460 cells. Ectopic expression of Wwox caused dramatic suppression of tumorigenicity of A549, H460, and H1299 cells in nude mice after Ad-WWOX infection and after ponA induction of Wwox expression in H1299 lung cancer cells. Tumorigenicity and in vitro growth of U2020 (Wwox-positive) lung cancer cells was unaffected by Wwox overexpression. This study confirms that WWOX is a tumor suppressor gene and is highly effective in preventing growth of lung cancer xenografts, whether introduced through viral infection or by induction of a silent WWOX transgene.

The fragile genes FHIT and WWOX are inactivated coordinately in invasive breast carcinoma.

FHIT and WWOX are a tumor suppressor and a candidate suppressor that encompass the FRA3B and FRA16D fragile sites at chromosomes 3p14.2 and 16q23.3–24.1, respectively. Reduced or absent Fhit expression has been reported in two‐thirds of invasive breast tumors in association with adverse prognostic factors. Loss of 16q has been reported frequently in low‐grade, invasive breast tumors.

Loss of WWOX Expression in Gastric Carcinoma

Purpose: WW domain-containing oxidoreductase (WWOX) is a tumor suppressor gene that maps to the common fragile site FRA16D on chromosome 16q23.3–24.1. To investigate the role of the WWOX gene in the development of gastric carcinoma, we examined a large series of primary adenocarcinomas and nine gastric cancer cell lines for the expression of Wwox. Experimental Design: Loss of heterozygosity, reverse-transcription-PCR, and immunohistochemistry were used to assess the role of WWOX in stomach cancer. A total of 81 primary gastric adenocarcinoma were analyzed. Results: Loss of heterozygosity was observed in 31% of the cases and loss of Wwox protein expression was found in 65% of gastric adenocarcinoma primary specimens and 33% of gastric cancer cell lines. In addition, we found a high correlation between Wwox and Fhit protein expression. Conclusions: Our results indicate that alterations of the WWOX gene may be involved quite frequently in gastric tumorigenesis. Our data could be used in future studies to develop diagnostic and targeted therapy of stomach cancer.

The Histidine Triad Superfamily of Nucleotide-Binding Proteins

Histidine triad (HIT) proteins were until recently a superfamily of proteins that shared only sequence motifs. Crystal structures of nucleotide‐bound forms of histidine triad nucleotide‐binding protein (Hint) demonstrated that the conserved residues in HIT proteins are responsible for their distinctive, dimeric, 10‐stranded half‐barrel structures that form two identical purine nucleotide‐binding sites. Hint‐related proteins, found in all forms of life, and fragile histidine triad (Fhit)‐related proteins, found in animals and fungi, represent the two main branches of the HIT superfamily. Hint homologs are intracellular receptors for purine mononucleotides whose cellular function remains elusive. Fhit homologs bind and cleave diadenosine polyphosphates (ApnA) such as ApppA and AppppA. Fhit‐ApnA complexes appear to function in a proapoptotic tumor suppression pathway in epithelial tissues. In invertebrates, Fhit homologs are encoded as fusion proteins with proteins related to plant and bacterial nitrilases that are candidate signaling partners in tumor suppression. J. Cell. Physiol. 181:179–187, 1999.

FHIT: from gene discovery to cancer treatment and prevention

Chromosomal abnormalities, including homozygous deletions and loss of heterozygosity, are among the most common features of human tumours. The short arm of human chromosome 3, particularly the region 3p14.2, is a major site of such rearrangements. The 3p14.2 region spans the most active common fragile site of the human genome, encompassing a familial-kidney-cancer-associated breakpoint and a papilloma virus integration site. 6 years ago, the FHIT gene was identified in this region. Subsequent studies have shown that FHIT is commonly the target of chromosomal aberrations involving the long arm of human chromosome 3 and is thereby inactivated in most of the common human malignant diseases, including cancers of the lung, oesophagus, stomach, breast, and kidney. During the past 5 years, evidence has accumulated in support of a tumour-suppressor function for FHIT. In this review, we describe the recent findings in the molecular biology of FHIT with particular focus on the opportunities for treatment and prevention of cancer that have emerged.

Structure and expression pattern of human ALR , a novel gene with strong homology to ALL-1 involved in acute leukemia and to Drosophila trithorax

The ALL-1 gene is involved in human acute leukemia through chromosome translocations or internal rearrangements. ALL-1 is the human homologue of Drosophila trithorax. The latter is a member of the trithorax group (trx-G) genes which together with the Polycomb group (Pc-G) genes act as positive and negative regulators, respectively, to determine the body structure of Drosophila. We have cloned a novel human gene, ALR, which encodes a gigantic 5262 amino acid long protein containing a SET domain, five PHD fingers, potential zinc fingers, and a very long run of glutamines interrupted by hydrophobic residues, mostly leucine. The SET motif, PDH fingers, zinc fingers and two other regions are most similar to domains of ALL-1 and TRX. The first two motifs are also found in other trx-G and Pc-G proteins. The ALR gene was mapped to chromosome band 12q12-13, adjacent to the VDR gene. This region is involved in duplications and translocations associated with cancer. The analysis of ALR expression showed that its ∼18u2009kb long mRNA is expressed, like ALL-1, in most adult tissues, including a variety of hematopoietic cells, with the exception of the liver. Whole mount in situ hybridization to early mouse embryos indicates expression in multiple tissues. Based on similarities in structure and expression pattern, ALR is likely to play a similar role to ALL-1 and trx, although its target genes have yet to be identified.

Concordant loss of fragile gene expression early in breast cancer development

The FHIT and WWOX genes encompass the FRA3B and FRA16D fragile sites at chromosomes 3p14.2 and 16q23.3, respectively. Reduced Fhit and Wwox expression has been reported in approximately two‐thirds of invasive breast tumors. Expression of these fragile gene products, as well as ErbB2 and p53, were evaluated immunohistochemically in 44 pure and 31 adjacent‐to‐invasive ductal carcinoma in‐situ (DCIS) cases. Reduced Fhit and Wwox expression were observed in (i) 70% and 68% of pure DCIS; (ii) 52% and 55% of DCIS adjacent‐to‐invasive tumor cases; and (iii) 20% and 50% of adjacent normal tissue in pure DCIS cases. Reduced Wwox expression in adjacent normal tissue was observed in 30% of cases in the DCIS adjacent‐to‐invasive group. Reduced Fhit and Wwox expression was observed in 61% of adjoining invasive tumors. In all normal, pure DCIS, and DCIS adjacent‐to‐invasive lesions, Fhit and Wwox expression was positively associated (Pu2003=u20030.034, Pu2003=u20030.042, Pu2003=u20030.004, respectively) and in the invasive component there was a positive trend toward association (Pu2003=u20030.075). Fhit and Wwox were more frequently reduced in high‐grade lesions in the DCIS adjacent‐to‐invasive (Pu2003=u20030.025, Pu2003=u20030.004, respectively). In the pure DCIS group, there was a statistically significant negative association between Fhit and ErbB2 expression in DCIS (Pu2003=u20030.035). In summary, reduced Fhit and Wwox expression in in‐situ breast cancer was associated, which may contribute to the high‐grade DCIS–invasive tumor pathway.