Involvement of cyclin D1 and E2F1 in intramedullary apoptosis in myelodysplastic syndromes.
An unusually high incidence of apoptosis in S-phase cells is characteristically found in the bone marrow (BM) of patients with myelodysplastic syndromes (MDS). Previously, E2F1, c-myc, and Cyclin D1 have been shown to bring about both S-phase changes and/or apoptotic changes. We have already found a stoichiometric imbalance between pRb and E2F1 causing deregulated E2F1 activity in these disorders. In the present study, we investigated the status of Cyclin D1 in relation to E2F1 and apoptosis in 19 patients with a confirmed diagnosis of MDS in comparison with 6 healthy donors. Cyclin D1 was localized immunohistochemically using a specific monoclonal antibody (1:150 dilution) in plastic-embedded BM sections. The nuclear localization of Cyclin D1 graded on a subjective rating scale of 0 (negligible staining) to 8+ (highest), demonstrated negligible levels in normal marrows (median 1+), and in 11/19 evaluable MDS marrows. In contrast, 8/19 MDS biopsies showed an almost four-fold increase in Cyclin D1 localization (p< or =0.001). A western blot analysis of E2F1 in corresponding bone marrow (BM) aspirate mononuclear cells (MNC) demonstrated that the MDS patients with elevated Cyclin D1 expression also had a significant increase in E2F1 protein (p< or =0.03). Additionally, these patients revealed higher levels of mRNA of one of the E2F1 transcriptional target genes, dihydrofolate reductase (DHFR, p=0.01). Subsequently, the relationship of Cyclin D1 with apoptosis was elucidated in a colocalization experiment in BM biopsy sections using immunohistochemistry for Cyclin D1 and in situ end labeling of DNA (ISEL) for apoptosis. The percentage of ISEL-positive apoptotic cells was several fold higher in MDS as compared to normal BMs (p=0.009). Interestingly, 7-41% (median 20%) of the apoptotic cells in different MDS BMs revealed co-localization of Cyclin D1 in their nucleus, whereas in normal BMs co-localization was virtually absent (p=0.008). Thus, it is possible that in a subset of MDS patients, apoptotic death of bone marrow cells may involve Cyclin D1/E2F1 pathway.
Saberwal
Gurveen
G
MDS Center and Section of Myeloid Diseases, Rush-Presbyterian-St. Luke's Medical Center, Rush University, Chicago, IL 60612, USA.
Broderick
Eileen
E
Janssen
Imke
I
Shetty
Vilasini
V
Alvi
Sairah
S
Lisak
Laurie
L
Venugopal
Parameswaran
P
Raza
Azra
A
Mundle
Suneel D
SD
D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal / Q000378:metabolism; D017209:Apoptosis; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000166:cytology; D018797:Cell Cycle Proteins; D002467:Cell Nucleus / Q000378:metabolism; D019938:Cyclin D1 / Q000096:biosynthesis*; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D009190:Myelodysplastic Syndromes / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase; D014157:Transcription Factors / Q000096:biosynthesis*; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D019938:Cyclin D1