Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-06-30
2003-04-15
Jones, W. Gary (Department: 1634)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S007200, C435S007230, C436S063000, C436S064000, C530S300000
Reexamination Certificate
active
06548650
ABSTRACT:
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
BACKGROUND OF THE INVENTION
Terminal differentiation in human melanoma cells correlates with temporal changes in the expression of specific target genes. To define those genes that may be critical for this process a subtraction hybridization approach was used. cDNA libraries were constructed from actively proliferating HO-1 human melanoma cells (driver cDNA library) and cultures treated for various time periods with the combination of recombinant human fibroblast interferon (IFN-&bgr;) and mezerein (MEZ) (temporally spaced tester cDNA library) that induces terminal differentiation (Jiang and Fisher, 1993). From these two cDNA libraries, an HO-1 IFN-&bgr;+MEZ temporally spaced subtracted (TSS) cDNA library was constructed. Random screening of this TSS cDNA library identifies cDNAs that display differential expression as a function of induction of growth arrest and terminal differentiation, called melanoma differentiation associated (mda) genes. In the present study the properties of the novel mda-9 gene were analyzed. This cDNA encodes a unique protein of 298 amino acids with a predicted size of 32 to ~34 kDa. Southern blotting analysis indicates that mda-9 is an evolutionary conserved gene. Tissue distribution analysis documents comparable expression in fifty human tissues, with slightly elevated expression in brain (putamen) and spleen (adult and fetal). Treatment of HO-1 human melanoma cells with IFN-&bgr;+MEZ results in a biphasic induction of mda-9 with maximum expression 8 and 12 h post-treatment and reduced expression at 24 h. In terminally differentiated and irreversibly growth arrested human melanoma cells, the level of mda-9 mRNA is reduced. The suppression in mda-9 expression is not simply a function of growth inhibition, since treatment of HO-1 cells with interferons, including IFN-&bgr;, leukocyte interferon (IFN-&agr;) or immune interferon (IFN-&ggr;), elevates mda-9 expression even though they suppress growth. These studies demonstrate that subtraction hybridization using temporally spaced RNA samples, resulting in a TSS cDNA library, can identify genes, such as mda-9, that are down-regulated during terminal cell differentiation in human melanoma cells. Further studies are necessary to define the precise role of mda-9 in the process of terminal differentiation.
Cancer is a progressive disease characterized by both qualitative and quantitative changes in the phenotypes of evolving tumor cells (1-5). Although cancer can develop as a consequence of single or multiple genetic alterations, a common theme in carcinogenesis involves abnormal programs of differentiation (6-10). Attempts to exploit this defective differentiation process in cancer cells has led to the development of a therapeutic approach called ‘differentiation therapy’ (6-11). This strategy is based on the use of single or multiple agents that induce cancer cells to become more differentiated with a concomitant reduction or loss of growth potential (6-12). In order to utilize differentiation therapy as an effective clinical tool, further research is necessary to identify agents capable of efficiently inducing terminal differentiation in cancer cells without inducing nonspecific toxicity in normal cells. Additionally, the identification of genes that correlate with and may mediate terminal cell differentiation would represent valuable reagents for defining the molecular basis of terminal cell differentiation, for direct cancer therapeutic applications and for screening compounds for potential use in differentiation therapy (6-12).
In cultured human melanoma cells, the combination of IFN-&bgr;+MEZ results in terminal cell differentiation and an irreversible loss of proliferative potential (11, 13, 14). In this model system, a single treatment for 24 h is sufficient to induce >95% terminal differentiation in cells subsequently grown for 72 h in the absence of inducers (14,15). The rapid induction of terminal differentiation in the vast majority of treated cancer cells makes this system amenable for defining those gene expression changes that occur during and that may mediate this process (11,12,16-19). To begin to address on a molecular level the question of growth control and terminal differentiation in human melanoma cells and to directly clone genes involved in these processes we developed and used an efficient subtraction hybridization protocol (16). This approach has resulted in the cloning of both known and novel cDNAs that are differentially regulated during growth suppression, reversible differentiation and terminal differentiation in human melanoma and other cancer cell types (16-20). The contents of U.S. Pat. No. 5,643,761, issued Jul. 1, 1997 to Fisher et al. entitled “Method for Generating a Subtracted cDNA Library and Uses of the Generated Library” and of International Application PCT/US94/12160 filed Oct. 24, 1994, entitled “Method for Generating a Subtracted cDNA Library and Uses of the Generated Library” which published May 4, 1995 as WO 95/11986 are hereby incorporated by reference.
In the present study, the properties of a novel mda-9 gene identified by subtraction hybridization were described. mda-9 is an evolutionary conserved gene that encodes a protein of ~32 to ~34 kDa without sequence homology to previously identified proteins. Expression of mda-9 is seen in fifty human tissues, with slightly elevated expression in brain (putamen) and spleen (adult and fetal). Induction of growth suppression and differentiation in human melanoma cells following exposure to IFN-&bgr;+MEZ results in a decrease in mda-9 expression. These studies provide additional support for the hypothesis that induction of terminal differentiation and irreversible growth arrest in human melanoma cells involves multiple gene expression changes, including increases as well as decreases in the expression of specific target genes.
SUMMARY OF THE INVENTION
This invention provides a method for producing a temporally spaced subtracted cDNA library comprising: a) isolating temporally spaced RNAs from cells; b) generating cDNA inserts from the RNAs isolated from step (a); c) producing a temporally spaced cDNA library having clones containing the cDNA inserts generated from step (b); d) producing double stranded cDNA inserts from the temporally spaced cDNA library; e) denaturing the double stranded cDNA inserts; f) contacting the denatured double stranded cDNA inserts produced in step (e) with single-stranded DNAs from another cDNA library under conditions permitting hybridization of the single-stranded DNAs and the double-stranded cDNA inserts; g) separating the hybridized cDNA inserts from the unhybridized inserts; h) generating a cDNA library of the unhybridized inserts, thereby generating a temporally spaced subtracted cDNA library.
This invention further provides a temporally spaced subtracted library generated by the method for producing a temporally spaced subtracted cDNA library comprising: a) isolating temporally spaced RNAs from cells; b) generating cDNA inserts from the RNAs isolated from step (a); c) producing a temporally spaced cDNA library having clones containing the cDNA inserts generated from step (b); d) producing double stranded cDNA inserts from the temporally spaced cDNA library; e) denaturing the double stranded cDNA inserts; f) contacting the denatured double stranded cDNA inserts produced in step (e) with single-stranded DNAs from another cDNA library under conditions permitting hybridization of the single-stranded DNAs and the double-stranded cDNA inserts; g) separating the hybridized cDNA inserts from the unhybridized inserts; h) generating a cDNA library of the unhybridized inserts, thereby ge
Baker & Botts LLP
Chakrabarti Arun Kr.
Jones W. Gary
Trustees of Columbia University in the City of New York
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