Expression profiling of apoptosis-related genes in megakaryocytes: BNIP3 is downregulated in primary myelofibrosis.
OBJECTIVE
In order to identify factors involved in the aberrantly regulated apoptosis of megakaryocytes in primary myelofibrosis (PMF), the mRNA expression of human megakaryocytes in situ was quantified by real-time polymerase chain reaction low-density arrays.


METHODS
The mRNA from 200 to 300 laser-microdissected megakaryocytes per case from PMF (n=22) and control (n=10) bone marrow was reverse-transcribed into cDNA by random priming and subsequently amplified by primer-specific cDNA amplification. The mRNA of corresponding total bone marrow cells was reverse-transcribed into cDNA without the following amplification. For relative mRNA quantification, custom-made TaqMan low-density arrays with a setup of 48 different genes were applied. In addition, methylation analysis and immunohistochemistry of a selected candidate gene were accomplished.


RESULTS
A trend toward an overall downregulation of apoptosis-associated genes could be observed in megakaryocytes, whereas the total bone marrow cellularity exhibited an overall upregulation of these factors. Among several candidates with statistically significant deregulation BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and protein kinase C beta1 were shown to be the most aberrantly expressed genes.


CONCLUSIONS
Apoptosis-related gene expression profiling of human megakaryocytes reveals a set of candidates, most notably BNIP3, indicating that the increase of megakaryocytes in myeloproliferative neoplasia might not only be the result of increased proliferation but also of disturbed apoptosis.
Theophile
Katharina
K
Institute of Pathology, Hannover Medical School, Hannover, Germany.
Hussein
Kais
K
Kreipe
Hans
H
Bock
Oliver
O
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis*; D049109:Cell Proliferation; D015536:Down-Regulation; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D008533:Megakaryocytes / Q000473:pathology*; D008565:Membrane Proteins / Q000096:biosynthesis*; D020411:Oligonucleotide Array Sequence Analysis; D055728:Primary Myelofibrosis / Q000378:metabolism* / Q000473:pathology; D011493:Protein Kinase C / Q000096:biosynthesis; D064546:Protein Kinase C beta; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D051017:Apoptosis Regulatory Proteins; C090121:BNIP3 protein, human; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011493:Protein Kinase C; D064546:Protein Kinase C beta