Sphingosine kinase and uses thereof

A technology of sphingosine kinase and dimethylsphingosine, applied in the field of treatment and/or preventive treatment of cancer, down-regulation of neoplastic cell growth, can solve the problem that has only just begun to be understood, and the signal has not been confirmed OK, etc.

Inactive Publication Date: 2003-08-13
MEDVET SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the precise nature of the cellular activity so regulated and the roles and mechanisms of action of sphingosine kinases in this reg...

Method used

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  • Sphingosine kinase and uses thereof
  • Sphingosine kinase and uses thereof
  • Sphingosine kinase and uses thereof

Examples

Experimental program
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Embodiment 1

[0129] Transfection and analysis of NIH 3T3 fibroblasts

[0130] To study the oncogenic role of SphK, untransformed NIH 3T3 fibroblasts were transfected with human SphK cDNA recently cloned in our laboratory (Piston et al., 2000). The pooled stable transformants (termed SK-3T3) were used to avoid phenotypic artifacts that might result from the selection and propagation of individual clones from single transfected cells. In the SK-3T3 cell bank, the SphK activity increased more than 600-fold compared with the empty vector-transfected NIH 3T3 cells (N3T3) (attached figure 1 a). Immunoblot analysis revealed a specific protein band with an apparent molecular weight consistent with the predicted size of FLAG-tagged human SphK detected only in the SK-3T3 cell pool but absent in N-3T3 cells (attached figure 1 c). Intracellular levels of SphK's direct product, S1P, were also increased 4-5-fold in SK-3T3 cells, suggesting that stable transfectants harbor constitutively activated Sph...

Embodiment 2

[0134] Transfection and analysis of oncogene-transformed cells

[0135] When NIH 3T3 cells were transfected with activated mutant Ras (V12-Ras), SphK activity was significantly increased by 178±22% compared with parental cells (attached image 3 c). In contrast, cells transfected with v-Src (attached image 3c) or dominant negative Ras (N17-Ras) did not alter SphK activity, suggesting a specific relevance of SphK in an oncogenic transformation. Furthermore, foci formation was reduced by 42 ± 4% in V12-Ras-transformed cells when cells were treated with DMS, but not altered in v-Src-transformed cells (attached image 3 d), thus demonstrating the important role of SphK in Ras conversion. The partial effect of DMS at the fully effective dose (2.5 μM) in SphK-transformed cells suggests the possibility of a functioning SphK-independent pathway (attached image 3 d). On the other hand, the inability of DMS to inhibit v-Src conversion rules out a non-specific effect of DMS or gen...

Embodiment 3

[0137] Analysis of tumorigenicity

[0138] The tumorigenicity was then directly tested in NIH 3T3 cells overexpressing SphK. When SphK-transfected NIH 3T3 cells from a pool of stable transformants or selected clones were injected subcutaneously into NOD / SCID mice, tumors became evident at the site of injection within 3-4 weeks (Table 4 and Supplementary Table 4). Figure 4 ). Mice injected with vector-transfected 3T3 cells did not induce tumors during the 10-week observation period. Histological appearance of tumor sections showed fibrosarcomatous morphology with many mitotic figures (attached Figure 4 ). Western blot analysis of tumor-derived extracts revealed high levels of FLAG-tagged proteins (attached Figure 4 ), thus indicating that the neoplastic cells retained and expressed the SphK transgene. Thus, tumors developed from injected SphK-transfected cells but not from autonomously transformed NIH 3T3. This is the first demonstration that the wild-type lipid kinase...

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Abstract

The present invention relates generally to a method of modulating the growth of cells and, more particularly, to a method of down-regulating the growth of neoplastic cells. The present invention is useful, interalia, in the therapeutic and/or prophylactic treatment of cancers such as, but not limited to, solid cancers such as cancers of the colon, stomach, lung, brain, bone, oesophagus, pancreas, breast, ovary or uterus.

Description

field of invention [0001] The present invention relates generally to methods of modulating cell growth, and more particularly, the present invention relates to methods of down-regulating neoplastic cell growth. The present invention is particularly useful in the therapeutic and / or prophylactic treatment of cancers such as but not limited to solid cancers such as colon, stomach, lung, brain, bone, esophagus, pancreas, breast, ovary or uterus . Background of the invention [0002] A bibliography of publications cited by the authors throughout this specification is collected at the end of this specification. [0003] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that such prior art forms part of the common general knowledge in Australia. [0004] Sphingosine kinases are key regulatory enzymes in diverse cellular responses. Sphingosine-1-phosphate is known to be an important second messenger in...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K31/13A61K31/133A61K35/14A61K38/00A61K45/00A61P35/00A61P43/00C12N5/07C12N5/077C12N5/09C12N5/10C12N9/12C12N15/54C12Q1/02C12Q1/48C12Q1/68
CPCC12N9/1205A61K38/00A61K31/13A61P35/00A61P43/00
Inventor M·韦达斯J·甘布尔夏朴王丽君
Owner MEDVET SCI
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