Detection of protein tyrosine-kinase (PTK) gene expression pattern in normal and malignant T lymphocytes by combined PTK-specific polymerase chain reaction and parallel denaturing gradient gel electrophoresis.
Protein tyrosine kinases (PTKs) control key functions of normal and malignant cells. Comparison of PTK gene expression among various cell populations may be achieved by amplification of the PTK cDNAs using degenerate primers which recognize two relatively invariable regions within their catalytic domain. This approach produces a mixture of PTK cDNA fragments with identical or very similar lengths which are difficult to separate by standard gel electrophoresis. These mixed products are then analyzed in a random fashion which leads to redundant cloning of some and potential omission of other PTKs. By using parallel denaturing gradient gel electrophoresis (DGGE) we have been able to separate the amplified PTK cDNAs derived from the same T-lymphocyte population and compare their expression between various types of normal and malignant T lymphocytes. One such PTK is the type I receptor for insulin-like growth factor, which we found to be preferentially expressed by neoplastic T cells on the both mRNA and protein levels. The combination of PCR which uses PTK-specific primers and parallel DGGE of the amplified PTK cDNAs may prove useful in studying mechanisms of cell activation and malignant transformation and in identifying targets for therapies based on selective inhibition of oncogenic PTKs.
Wang
Zhi-Yong
ZY
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Zhang
Qian
Q
Wilson
John
J
Ratajczak
Mariusz Z
MZ
Wasik
Mariusz A
MA
D015152:Blotting, Northern; D020134:Catalytic Domain; D002455:Cell Division; D002469:Cell Separation; D002471:Cell Transformation, Neoplastic; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis* / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013601:T-Lymphocytes / Q000201:enzymology*
D018076:DNA, Complementary; D012333:RNA, Messenger; D011505:Protein-Tyrosine Kinases