Yonsei Med J.  2008 Apr;49(2):279-287. 10.3349/ymj.2008.49.2.279.

Secretion of Biologically Active Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor by Transduced Gastric Cancer Cells

• Kim HY
• Park GS
• Shin HJ
• Park S
• Kim K
• Kim HI

• KMID: 1084502
• DOI: http://doi.org/10.3349/ymj.2008.49.2.279

Abstract

PURPOSE
Gastric cancer has the highest incidence rate among cancers in Asia. The advanced type of signet ring cell carcinoma has poor prognosis compared to other types of gastric cancer. The immuno-gene therapy with cytokine-based tumor vaccines has not yet been investigated for gastric cancer. The granulocyte macrophage colony-stimulating factor (GM-CSF)-based tumor vaccine has been demonstrated as the most potent stimulator for specific and long-lasting systemic tumor immunity. MATERIALS AND METHODS: In the present study, KATO III cells, the human signet ring cell gastric carcinoma cell line, were genetically modified by the transduction with the human GM-CSF cDNA or the modified hGM-CSF in replication-deficient retroviruses. The genomic integrations and mRNA expressions of the transgenes were determined by Southern and Northern blot analyses. RESULTS: Wild type (wt) or modified hGM-CSF was integrated into the genome of KATO III cells. The modified hGM-CSF mRNA was more stable than that of wt. The KATO III cells with the modified hGM-CSF produced higher level of hGM-CSF (12.4-19 ng/10(6)cells/48hrs) than that with wt hGM-CSF, when determined by enzyme-linked immunosorbent assay (ELISA). The secreted recombinant hGM-CSF could support the proliferation of the GM-CSF-dependent cell line, indicating that the hGM-CSF secreted by the transduced KATO III cells has biological activities. Irradiated, transduced KATO III cells continued to secret hGM-CSF without proliferation. CONCLUSION: Our results suggest that GM-CSF secreting KATO III cells could be tested for the treatment of gastric cancer as an allogeneic tumor vaccine as a part of immunotherapeutic treatment.

Keyword

Human granulocyte macrophage colony-stimulating factor transduced gastric cancer cell line; allogeneic gastric cancer cell vaccine; immuno-gene therapy

MeSH Terms

Base Sequence
Blotting, Northern
Blotting, Southern
Cell Line, Tumor
Enzyme-Linked Immunosorbent Assay
Granulocyte Macrophage Colony-Stimulating Factors,
Humans
Mutagenesis
RNA, Messenger/genetics/metabolism
Recombinant Proteins/metabolism/*secretion
Stomach Neoplasms/genetics/metabolism/pathology
Transduction, Genetic

Figure

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  Fig. 1 Targeted mutagenesis of human GM-CSF sequence. To increase the hGM-CSF expressions, both the 5'-end start codon flanking sequences (A) and 3'-end UTR (B) were mutated using recombinant PCR techniques. (A) The suboptimal AUG start codon flanking sequences were replaced to a part of Kozak sequences. (B) AUUUA repeat sequences in 3'-end UTR of the hGM-CSF gene were mutated to AUGUA repeat sequences (nts 672-688) and deleted (nts 647-671 and 689-711).

• 

  Fig. 2 Chromosomal integrations of the wt and modified hGM-CSF transgenes in the transduced KATO III cells. Southern blot analysis was performed to detect the integrated wt and modified hGM-CSF transgenes in the respective transduced KATO III cells. Chromosomal DNA was isolated from two representative clones of LGMIN- and LGMHIN-transduced KATO-III cells. Thirty µg of Xba I-digested chromosomal DNA was resolved in 0.8% agarose gels and transferred to nylon membrane. The DNA on the membrane was hybridized with random-primed 32P-labeled GM-CSF specific probe and subjected to autoradiography.

• 

  Fig. 3 The hGM-CSF mRNA expressions in the wt and modified hGM-CSF transduced KATO III cells. Northern blot analysis was performed to detect the wt and modified hGM-CSF mRNAs in the respective transduced KATO III cells. Total RNA was isolated from four representative clones of LGMIN- and LGMHIN-transduced KATO III cells. Transduced KATO III cells with LGFPIN, GFP-containing retrovirus, were used as a negative control. Twenty-five µg of total RNA was denatured and electrophoresed through 1% agarose gel containing formaldehyde and transferred to nylon membrane. The GM-CSF mRNA on the membrane was hybridized with random-primed 32P-labeled GM-CSF specific probe and subjected to autoradiography. 18S ribosomal RNAs are indicated as a loading control.

• 

  Fig. 4 The stabilities of wt and modified hGM-CSF mRNAs in the wt and modified hGM-CSF transduced KATO III cells. Northern blot analysis was performed to compare the mRNA stabilities of wt and modified hGM-CSF mRNAs. LGFPIN transduced KATO III cells were used as a negative control. Two representative clones of LGMIN and LGMHIN transduced KATO III cells were treated with 2.5 mg/mL of actinomycin-D for 3 hrs, and total RNA was isolated. Twenty-five µg of total RNA was denatured and electrophoresed through 1% agarose gel containing formaldehyde, and blotted onto nylon membrane. GM-CSF mRNA on the membrane was hybridized with random-primed 32P-labeled GM-CSF-specific probe and subjected to autoradiography. 0.9kbs GM-CSF mRNAs from LGMIN and LGMHIN transduced KATO III cells are indicated as squares and asterisks, respectively. 18S ribosomal RNAs are indicated as a loading control.

• 

  Fig. 5 Proliferation of GM-CSF dependent leukemic cells by the addition of recombinant hGM-CSF from LGMHIN transduced KATO III cells. To examine biological activity of the recombinant hGM-CSF, culture supernatant from a clone 18 of LGMHIN-transduced KATO III cells was added to AML-193 cells (GM-CSF dependent leukemic cell line) and proliferation of AML-193 cells was monitored by 3H-thymidine uptake.

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