Stably Transfected Antisense Granzyme B and Perforin Constructs Inhibit Human Granule-Mediated Lytic Ability

Markian R. Bochan, W. Goebel, Zacharie Brahmi

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Abstract

In human NK cells and CTL it has been shown that release of lytic molecules is, at least in part, responsible for the lysis of target cells (TC). Of the various types of molecules thought to be involved in cell-mediated cytotoxicity (CMC), perforin and the serine proteases (granzymes A and B) are the best described. Using mammalian expression vectors (pRSV-neo and pSV2-neo), antisense constructs for perforin and granzyme B were independently electroporated into YT-INDY, a human non-MHC-restricted, IL-2-independent, cytotoxic lymphocyte. Transfected YT-INDY was then selected for expression of the plasmid by antibiotic G418 resistance. The presence of plasmid was confirmed by detection of the integrated plasmid G418 resistance gene using PCR. The presence of antisense perforin in YT-INDY (YT-xPFP) inhibited lytic ability by >95% compared to YT-INDY transfected with plasmid alone or plasmid with unrelated antisense (YT-neo, YT-ctrl, respectively). Likewise, the presence of antisense GrB (YT-xGrB) inhibited the lytic ability of YT-INDY by >95%. Western analysis revealed a 30% decrease in the level of perlatin and a 55% decrease in granzyme B protein levels compared to YT-neo. Northern analysis using oligo probes complementary to perforin and granzyme B mRNA showed a decrease in their respective message levels. In conclusion, stably transfected antisense constructs for perforin and granzyme B essentially eliminated the lytic ability of YT-INDY. These results strongly indicate that both perforin and granzyme B are required by this human cytotoxic lymphocyte for effective TC lysis.

Original languageEnglish
Pages (from-to)234-239
Number of pages6
JournalCellular Immunology
Volume164
Issue number2
DOIs
StatePublished - Sep 1995

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Plasmids
Granzymes
Perforin
Lymphocytes
Serine Proteases
Microbial Drug Resistance
Natural Killer Cells
Interleukin-2
perforin-granzyme B
Polymerase Chain Reaction
Messenger RNA
Genes
Proteins
antibiotic G 418

ASJC Scopus subject areas

  • Immunology
  • Cell Biology

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Stably Transfected Antisense Granzyme B and Perforin Constructs Inhibit Human Granule-Mediated Lytic Ability. / Bochan, Markian R.; Goebel, W.; Brahmi, Zacharie.

In: Cellular Immunology, Vol. 164, No. 2, 09.1995, p. 234-239.

Research output: Contribution to journalArticle

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abstract = "In human NK cells and CTL it has been shown that release of lytic molecules is, at least in part, responsible for the lysis of target cells (TC). Of the various types of molecules thought to be involved in cell-mediated cytotoxicity (CMC), perforin and the serine proteases (granzymes A and B) are the best described. Using mammalian expression vectors (pRSV-neo and pSV2-neo), antisense constructs for perforin and granzyme B were independently electroporated into YT-INDY, a human non-MHC-restricted, IL-2-independent, cytotoxic lymphocyte. Transfected YT-INDY was then selected for expression of the plasmid by antibiotic G418 resistance. The presence of plasmid was confirmed by detection of the integrated plasmid G418 resistance gene using PCR. The presence of antisense perforin in YT-INDY (YT-xPFP) inhibited lytic ability by >95{\%} compared to YT-INDY transfected with plasmid alone or plasmid with unrelated antisense (YT-neo, YT-ctrl, respectively). Likewise, the presence of antisense GrB (YT-xGrB) inhibited the lytic ability of YT-INDY by >95{\%}. Western analysis revealed a 30{\%} decrease in the level of perlatin and a 55{\%} decrease in granzyme B protein levels compared to YT-neo. Northern analysis using oligo probes complementary to perforin and granzyme B mRNA showed a decrease in their respective message levels. In conclusion, stably transfected antisense constructs for perforin and granzyme B essentially eliminated the lytic ability of YT-INDY. These results strongly indicate that both perforin and granzyme B are required by this human cytotoxic lymphocyte for effective TC lysis.",
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