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Author(s): M. Hattori [1]; A. Fujiyama [1]; T. D. Taylor [1]; H. Watanabe [1]; T. Yada [1]; H.-S. Park [1]; A. Toyoda [1]; K. Ishii [1]; Y. Totoki [1]; D.-K. [...]

Author(s): The chromosome 21 mapping and sequencing consortium; M. Hattori; A. Fujiyama; T. D. Taylor; H. Watanabe; T. Yada; H.-S. Park; A. Toyoda; K. Ishii; Y. Totoki; D.-K. Choi; Y. [...]

Inhibitor of differentiation/DNA binding (Id) proteins comprise a class of helix-loop-helix transcription factors involved in proliferation, differentiation, apoptosis, and carcinogenesis. We have shown that while Id2 is induced by UVB in primary keratinocytes, Id3 is upregulated only in immortalized cells. We have now determined that the consequences of ectopic expression of Id3 protein are strikingly different between immortalized and primary keratinocytes. Overexpression of Id3 induces a significant increase in apoptotic cells as revealed by Annexin V positivity as well as proteolytic processing of caspase-3 in immortalized, but not in primary keratinocytes. Id3-green fluorescent protein (GFP)-positive cells exhibited a fivefold increase in apoptotic nuclear fragmentation compared to Id3-GFP-negative cells. These apoptotic responses were accompanied by activation of caspase-3, as shown by immunocytochemical staining with antibodies to active caspase-3. Immunostaining with antibodies to the active form of caspase-9 as well as to the active form of Bax further revealed that induction of apoptosis in Id3-overexpressing keratinocytes occurred via a mitochondrial-caspase-9-mediated pathway. Coexpression of dominant-negative caspase-9 with Id3 significantly suppressed apoptotic nuclear fragmentation, indicating that caspase-9 activation is essential for Id3-induced cell death. This response was also markedly attenuated by coexpression with the Bax antagonist antiapoptotic protein Bcl2, confirming a role for Bax activation in this apoptotic response. Id3-induced Bax activation may result from increased expression of Bax protein. Furthermore, reduction of Id3 expression by small interfering RNAs abrogated the UVB-induced proteolytic activation of caspase-3 in these cells. These data together suggest that UVB-induced apoptosis of immortalized keratinocytes is at least in part due to Id3 upregulation in these cells.

In order to clone candidate tumor suppressor genes whose loss contributes to the pathogenesis of neuroblastoma (NB), we performed polymerase chain reaction (PCR) screening using a high-density sequence tagged site-content map within a commonly deleted region (chromosome band 1p36) in 24 NB cell lines. We found a approximately 480 kb homozygously deleted region at chromosome band 1p36.2 in one of the 24 NB cell lines, NB-1, and cloned the human homologue (KIF1B-beta) of the mouseKif1B-beta gene in this region. The KIF1B-beta gene had at least 47 exons, all of which had a classic exon-intron boundary structure. Mouse Kif1B is a microtubule-based putative anterograde motor protein for the transport of mitochondria in neural cells. We performed mutational analysis of the KIF1B-beta gene in 23 cell lines using 46 sets of primers and also an allelic imbalance (AI) analysis of KIF1B-beta in 50 fresh NB samples. A missense mutation at codon 1554, GTG (Gly) to ATG (Met), silent mutations at codon 409 (ACG to ACA) and codon 1721 (ACC to ACT), and polymorphisms at codon 170, GAT (Asp) to GAA (Glu), and at codon 1087, TAT (Tyr), to TGT (Cys), were all identified, although their functional significances remain to be determined. The AI for KIF1B-beta was slightly higher (38%) than those for the other two markers (D1S244, D1S1350) (35 and 32%) within the commonly deleted region (1p36). Reverse transcriptase-PCR analysis of the KIF1B-beta gene revealed obvious expression in all NB cell lines except NB-1, although decreased expression of the KIF1B-beta gene was found in a subset of early- and advanced-stage NBs. These results suggest that the KIF1B-beta gene may not be a candidate for tumor suppressor gene of NB.

Loss of heterozygosity of the distal region of chromosome 1p where tumor suppressor gene(s) might harbor is frequently observed in many human cancers including neuroblastoma (NBL) with MYCN amplification and poor prognosis. We have identified for the first time a homozygously deleted region at the marker D1S244 within the smallest region of overlap at 1p36.2-p36.3 in two NBL cell lines, NB-1 and NB-C201 (MASS-NB-SCH1), although our genotyping has suggested the possibility that both lines are derived from the same origin. The 800-kb PAC contig covering the entire region of homozygous deletion was made and partially sequenced (about 60%). The estimated length of the deleted region was 500 kb. We have, thus far, identified six genes within the region which include three known genes (DFF45, PGD, and CORT) as well as three other genes which have been reported during processing our present project for the last 3(1/2) years (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14). They include the genes related to apoptosis, glucose metabolism, ubiquitin-proteasome pathway, a neuronal microtubule-associated motor molecule and biogenesis of peroxisome. At least three genes (HDNB1/UFD2, KIAA0591F/KIF1B-beta, and PEX14) were differentially expressed at high levels in favorable and at low levels in unfavorable subsets of primary neuroblastoma. Since the 1p distal region is reported to be imprinted, those differentially expressed genes could be the new members of the candidate NBL suppressor, although RT-PCR-SSCP analysis has demonstrated infrequent mutation of the genes so far identified. Full-sequencing and gene prediction for the region of homozygous deletion would elucidate more detailed structure of this region and might lead to discovery of additional candidate genes. Oncogene (2000) 19, 4302 - 4307

Properties of a virus-like artificial gene delivery vehicle, synthesised from recombinant major coat protein of mouse polyoma virus, have been explored. The protein, VP1, self assembles into protein spheres, or 'pseudocapsids, which can bind and transfer DNA into cells in vitro and in vivo. Here, the ability of pseudocapsids to carry DNA into a complex cell system (ex vivo organ cultures of rabbit cornea) or whole animals (mice) has been assessed. Evidence from histochemical and PCR experiments indicate that pseudocapsids stimulate uptake and stable maintenance of marker DNA in nondividing corneal cells as efficiently as a recombinant adenovirus. In athymic and immunocompetent mice, gene transmission occurs with no apparent adverse effects on the animals. In the presence of pseudocapsids, the marker gene was transferred to a range of organs, including the brains of animals, following peripheral or intranasal administration. In immunocompetent mice, significant long-term transcriptional expression (at least 22 weeks) was observed with pseudocapsids, a period significantly longer than observed with DNA alone (several weeks only), again with no obvious adverse effects. This study demonstrates that pseudocapsids from the murine virus, polyoma, constitute a novel transfer agent for long-term gene therapeutic applications in tissues or whole animals.

Gene transfer to provide long-term expression of a therapeutic product, without introducing unwelcome genetic information, is a goal being sought for therapy of both hereditary and acquired diseases. Polyoma virus pseudocapsids, generated from a VP1-expressing recombinant baculovirus, lack viral DNA and have been successfully used to introduce small exogenous genes stably into cells in vitro by a process designated 'pseudofection'; although pseudocapsids protect only about 3 kbp of exogenous DNA, low efficiency transfer of a larger fragment (6.2 kbp) has been observed. Here, expression of a 7.2 kbp plasmid (pCMV beta) encoding the beta-galactosidase gene was assessed to monitor not only efficiency, but the ability of pseudocapsids to transfer larger-sized DNA on their own, or in the presence of the polycation, poly-L-lysine, added to protect nonencapsidated DNA. When complexed to pseudocapsids only, the efficiency of expression of the transferred beta-galactosidase gene (in human or rodent cells), although low, appeared to stabilise with time. In the presence of polylysine, unencapsidated DNA was shown to be protected against DNase activity, but electron microscopy (EM) revealed the formation of large mixed aggregates. The addition of pseudocapsids to these aggregates, and measurement of mobilities of the complexes in CsCl equilibrum centrifugation, indicated that they contained negligible amounts of VP1. For subsequent pseudofection experiments, DNA was complexed first with pseudocapsids, then polylysine was added. The latter did not appear to displace pseudocapsids from DNA, and was found to increase the efficiency of short-term expression both in in vitro and in vivo experiments. Gene expression, analysed histochemically or by the polymerase chain reaction, revealed transcriptional activity of the input gene, with expression first diminishing, then stabilising over time. The presence of pseudocapsids, in complexes with DNA with or without polylysine, allowed for stable and persistent gene expression.

An automated DNA sequencing system, the collaborative effort of Japanese scientists and engineers, which has a potential output of up to 108,000 bases per day is described.

The complete DNA sequence of the A2 strain of polyoma virus has been determined. It consists of 5,292 base pairs. The sequence is analysed in terms of its coding potential and sites of possible functional significance or structural interest. The polyoma virus genome is compared with those of related tumour viruses, simian virus 40 and BK virus.