**6. Survivin splice variants**

192 Advances in Cancer Therapy

Functional annotation analysis indicates that genes related with dorso-ventral axis formation or epidermal growth factor receptor signaling pathway (EGFR) are significantly enriched in the shared genes associated with LSC and Survivin signaling in the KEGG database (http://www.genome.jp/kegg/) (P<0.03). Similarly, genes associated with regulation of EGFR signaling pathway are enriched in the Gene Ontology database (P<0.02). Our analysis shows that molecules shared by LSC and Survivin signaling are mapped on a functional signaling network that connects through the EGFR pathway (Figure 4). EGFR signaling

**5. Survivin modulates gene expression in LSC that connects through an** 

Fig. 4. Genes regulated by Survivin in ITD-Flt3+ KSL cells and deregulated in LSC are mapped on a functional signaling network associated with the epidermal growth factor receptor signaling pathway. Epidermal growth factor receptor signaling pathway (EGFR) is significantly enriched in the shared genes associated with LSC and Survivin signaling by pathway enrichment analysis in KEGG and Gene Ontology databases. Our analysis shows that 13 molecules shared by LSC and Survivin signaling are mapped on a functional signaling network associated with EGFR. The network was created based on the KEGG database using Cytoscape software. Gray and black circle represents down regulation and up regulation by Survivin gene deletion, respectively. The hatched circle

**EGFR signaling pathway** 

shows EGFR.

Survivin, like many genes, is alternatively spliced and variants having both similar or opposite activities have been identified, some of which are of prognostic significance (Fukuda & Pelus, 2006; Sampath & Pelus, 2007) . Spliced variants identified to date include Survivin 2 (Caldas et al., 2005; Mahotka et al., 2002) , 2B, Ex3 (Ling et al., 2005; Mahotka et al., 1999; Mahotka et al., 2002; Song & Wu, 2005) and 3B (Badran et al., 2004), and some of these variants have been evaluated for their anti- or pro-apoptotic function and ability to interact with wild-type Survivin and other chromosomal passenger proteins (CPC) in some model systems. More recently, several new Survivin splice variants were found in the expressed sequence tag (est) database or described based on RT-PCR using RNA from Molt-4 cells (Li & Ling, 2006; Mola et al., 2007), however the biological functions of these new variants are not known. Overall, with most all the variants reported to date, there is still a considerable lack of understanding of their biology and their function and prognostic value is only now being realized (Sampath & Pelus, 2007). In order to better understand the biology of human Survivin splice variants, we cloned each variant and evaluated their antiapoptotic functions. In the process, we identified a new splice variant we termed Survivin 3 that is similar to Survivin 3B (Figure 5). To determine if this was truly a novel variant, a forward primer specific for all splice variants and a splice variant specific reverse primer were designed and used to amplify cDNA using a polydT reverse transcribed cDNA library from HL60, K562 and Mo7e-Bcr-abl cells, the products resolved on an agarose gel and ~400bp bands excised, cloned and sequenced. Sequence analysis confirmed the presence of the novel splice variant. Survivin 3 contains 12 new amino acids at the Cterminus compared to wild-type Survivin and differs from all of the previously reported Survivin splice variants. Secondary structure prediction using Jpred suggests that Survivin 3 is structurally similar to wild-type Survivin, with the new amino acids predicted to be capable of forming a C-terminal helix that could potentially form the coiled coil domain, even though the length of this helix would be smaller than wild-type Survivin.
