Cancer Res Treat.  2004 Aug;36(4):246-254.

Identification of Genes Involved in Liver Cancer Cell Growth Using an Antisense Library of Phage Genomic DNA

• Lee YH
• Kim YH
• Park JG

Abstract

PURPOSE
Genes involved in liver cancer cell growth have been identified using an antisense library of large circular (LC-) genomic DNA of a recombinant M13 phage. MATERIALS AND METHODS: A subtracted cDNA library was constructed by combining procedures of suppression subtractive hybridization (SSH) and unidirectional cloning of the subtracted cDNA into an M13 phagemid vector. Utilizing the life cycle of M13 bacteriophages, LC-antisense molecules derived from 1, 200 random cDNA clones selected by size were prepared from the culture supernatant of bacterial transformants. The antisense molecules were arrayed for transfection on 96-well plates preseeded with HepG2. RESULTS: When examined for growth inhibition after antisense transfection, 153 out of 1, 200 LC-antisense molecules showed varying degrees of growth inhibitory effect to HepG2 cells. Sequence comparison of the 153 clones identified 58 unique genes. The observations were further extended by other cell-based assays. CONCLUSION: These results suggest that the LC-antisense library offers potential for unique high-throughput screening to find genes involved in a specific biological function, and may prove to be an effective target validation system for gene-based drug discovery.

Keyword

Functional genomics; Recombinant M13 phage; LC-antisense library; Liver cancer; Target validation

MeSH Terms

Bacteriophage M13
Bacteriophages*
Clone Cells
Cloning, Organism
DNA*
DNA, Complementary
Drug Discovery
Gene Library
Hep G2 Cells
Life Cycle Stages
Liver Neoplasms*
Liver*
Mass Screening
Transfection
DNA
DNA, Complementary

Figure

• 

  Fig. 1 A schematic diagram for the construction of a random gene LC-antisense library. Total RNA was prepared from a pair of liver normal and cancer tissues, with both mRNA populations converted into cDNA. The tester cDNA (from cancer tissue) and driver cDNA (from normal tissue) were digested with RsaI to obtain shorter and blunt-ended fragments. Two hybridization reactions were performed, followed by suppression PCR to selectively amplify sequences overexpressed or expressed only in liver cancer cells, but not in normal liver cells. The subtracted cDNA pool was cloned into the multicloning site of the pBS SK (-) vector in the same direction as the LacZ gene. The cDNA plasmid library was transformed into E.coli competent cells. LC-antisense molecules were prepared on a large scale by superinfecting competent bacterial cells with a helper phage, and arrayed in 96-well plates.

• 

  Fig. 2 Microscopic observations after LC-antisense transfection to HepG2 cells. Growth Inhibitions of HepG2 cells were examined by light microscopy 4 days post transfection of the LC-antisense library (×200). (A)-(C); control treatments as indicated, (D)-(I); HepG2 cells treated with six different LC-antisense molecules. In this figure, the data acquired from the treatments with 6 out of 1,200 LC-antisense types are representatively shown as an example. *Li., liposome

• 

  Fig. 3 Examination for growth inhibition by an MTT reduction assay. LC-antisense species of 58 genes were transfected into HepG2 cells. Sham treated cells with liposome alone or with control DNA+liposome complexes were simultaneously assayed.

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  Fig. 4 Effects of LC-antisense molecules on the cell cycle progression. The LC-antisense molecules of 58 genes were transfected into HepG2 cells along with the controls. Cells were harvested 48 h post transfection. Functional analysis was perfomed on an equal number of cells (104 events) by flow cytometry after DNA staining with propidium iodide. In this figure, the data acquired from the treatments with 6 out of 58 LC-antisense types are representatively shown as an example. (A)-(C) control treatments as indicated; (D) LCAS 11; (E) LCAS 14; (F)LCAS 15; (G) LCAS 16; (H) LCAS 29; (I) LCAS 31. *Li., liposome

• 

  Fig. 5 Induction of apoptotic DNA ladder formation by LC-antisense molecules. The LC-antisense molecules of 58 genes were transfected into HepG2 cells along with the controls. Genomic DNA was extracted 48 h post transfection and run on a 1.6% agarose gel. In this figure, the data acquired from treatments with 6 out of 58 LC-antisense types are representatively shown as an example. Lane M, 100 bp DNA ladder size marker; lane 1, sham treatment; lane 2, control DNA+liposome complexes; lane 3, LCAS 11; lane 4, LCAS 14; lane 5, LCAS 15; lane 6, LCAS 16; lane 7, LCAS 21; lane 8, LCAS 29; lane 9, cisplatin (positive control).

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