JAK2V617F and p53 mutations coexist in erythroleukemia and megakaryoblastic leukemic cell lines
© Zhao et al.; licensee BioMed Central Ltd. 2012
Received: 18 May 2012
Accepted: 21 June 2012
Published: 21 June 2012
JAK2V617F, a gain-of-function mutant form of tyrosine kinase JAK2, is found in the majority of patients with Ph- myeloproliferative neoplasms (MPNs), a group of chronic hematological diseases that often lead to acute leukemia. The current study is intended to find other gene mutations that collaborate with JAK2V617F to cause leukemic transformation.
Total RNA and genomic DNA were isolated from two JAK2V617F-positive cell lines, namely, erythroleukemic HEL and megakaryoblastic leukemic SET-2 cells. Candidate genes were amplified by PCR and further sequenced.
Homozygous mutations of the TP53 gene which encodes tumor suppressor p53 were found in HEL and SET-2 cells. While HEL cells, which have homozygous JAK2V617F, contain a rare M133K p53 mutation, SET-2 cells, which have a heterozygous JAK2V617F mutation, contain a common R248W p53 alteration. Western blot analyses revealed high levels of p53 expression in both cells. M133K and R248W are located in the DNA binding domain of p53. Structural analyses revealed that they potentially disrupt the interaction of p53 with DNA, thereby causing loss of p53 function.
JAK2V617F and p53 mutations coexist in leukemia cells. We believe that JAK2V617F is able to drive leukemic transformation when the function of tumor suppressor p53 is lost. The interplay of JAK2V617F with p53 may affect the progression of MPNs.
KeywordsJAK2 TP53 Leukemia Mutations Transformation
Ph- myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders in which one or more myeloid lineages are abnormally amplified. These diseases represent a group of chronic conditions including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) . MPNs mainly affect older people with an average age of onset of 55 years. Complications associated with MPNs include development of acute leukemia as well as thrombosis, hemorrhage, and myeloid metaplasia. The major molecular lesion in these diseases is JAK2V617F, which occurs in over 90% of PV and over 50% of ET and PMF [1, 2]. JAK2V617F has enhanced tyrosine kinase activity, and it causes constitutive activation of down-stream signal transducers when expressed in cells . Studies have demonstrated that transgenic expression or knock-in of JAK2V617F in mice causes MPN-like phenotypes [4, 5]. However, whether or not JAK2V617F is able to drive leukemic transformation is not known.
Malignant transformation usually involves a gain-of-function mutation of oncogenes and a loss-of-function mutation of tumor suppressor genes. Among various tumor suppressors, p53, which is encoded by the TP53 gene, has been extensively studied [6–8]. TP53 is mutated or inactivated in over 60% of cancers. Normal p53 suppresses malignant transformation by controlling cell cycle progression, ensuring the fidelity of DNA replication and chromosomal segregation, and inducing apoptosis in response to potentially deleterious events. Interestingly, mutations of p53 are the most common in solid tumors but relatively rare in blood cell malignancies such as leukemia [7, 9]. In this study, we investigated the mutation status of p53 in two JAK2V617F-positive leukemic cell lines. We found mutations of p53 in both cells. One cell line bears a rare M133K mutation, while the other contains a R248W alteration frequently found in other tumors.
Results and discussion
Identification of p53 mutations in HEL and SET-2 cells
Structural analysis of p53 mutants found in HEL and SET-2 cells
The mutation rate of p53 in leukemia overall is very low, but it is much higher in acute myeloid leukemia with a complex aberrant karyotype [7, 9, 16]. Interestingly, both HEL and SET-2 cells display an abnormal karyotype with selective JAK2 chromosomal amplification and concomitant deletion of the residual homolog . Indeed, the 6:1 JAK2V617F/JAK2 ratio found in our current study suggests amplification of the mutant JAK2 allele. In addition, earlier studies identified p53 mutations in about half of MPN patients in blast crisis [17, 18], suggesting inactivation of p53 may be relatively frequent in blastic transformation of MPNs and is required for the process. These studies were conducted before the discovery of JAK2V617F but included analysis of NRAS and KRAS which were found intact. Our current study demonstrates coexistence of JAK2V617F and mutations of p53 in leukemia cells. This provides evidence that JAK2V617F likely drives leukemic transformation when p53 function is lost. We also believe that the dependence of MPN phenotypes on aging may be related to decreasing activity of p53. Among various factors affecting aging, p53 is most extensively studied [19–21]. In fact, loss of p53 activity is considered an aging process. Besides p53 mutations, p53 function can also be inactivated by other mechanisms. For example, a recent study demonstrated that JAK2V617F negatively regulates p53 stabilization by enhancing MDM2 via La expression in MPNs . Our data further support the notion that JAK2V617F interacts with p53 to promote progression of MPNs. Therefore, inhibiting JAK2V617F and maintaining p53 function are of utmost therapeutic importance.
We identified the coexistence of JAK2V617F and p53 mutations in leukemia cells. This suggests that JAK2V617F is able to drive leukemic transformation when the tumor suppressor function of p53 is lost. Decreased p53 activity as a consequence of aging may also affect the progression of MPNs. Understanding the interplay of JAK2V617F and p53 should have major implications for prevention and treatment of the diseases.
Cells and DNAs
HEL (ATCC no. TIB-180) and SET-2 (DSMZ no. ACC 608) cell lines were purchased from American Type Culture Collection ( ATCC) and German Collection of Microorganisms and Cell Cultures (DSMZ), respectively. The cells were maintained in RPMI 1640 medium supplemented with 20% heat-inactivated fetal bovine serum. White blood cells were isolated from de-identified normal blood samples upon lysis of red blood cells. Total RNAs were isolated from cells by using the Trizol reagent (Invitrogen), and single strand cDNAs were synthesized with random primers by using a reverse transcription kit from Promega. Genomic DNAs were purified by using the phenol/chloroform method after proteinase K digestion of whole cell lysates.
PCR and sequencing analysis
PCR was run with high fidelity Phusion DNA polymerase. The entire coding regions of p53 and JAK2 were amplified from single strand cDNAs. The PCR primers used were GCCAGACTGCCTTCCGGGTCACT (forward) and AGAGATGGGGGTGGGAGGCTGTC (reverse) for p53, and TGCATGGGAATGGCCTGCCTTAC (forward) and CTTTCATCCAGCCATGTTATCCCTTA (reverse) for JAK2. Genomic DNA surrounding the entire exon 14 of the JAK2 gene was amplified by PCR from genomic DNAs with primers GATCTCCATATTCCAGGCTTACACA (forward) and TATTGTTTGGGCATTGTAACCTTCT (reverse). The PCR products were gel-purified and subjected to DNA sequencing analyses by using an ABI 3730 capillary sequencer at the core facility of University of Oklahoma Health Sciences Center. DNA sequencings were performed from both forward and reserve directions. To determine the ratio of JAK2 and JAK2V617F at the genomic or cDNA level, real time PCR analyses were performed as previously described , except that purified plasmid DNAs were used as standards .
Western blot analyses
Cells were extracted in SDS gel sample buffer. Upon separation on 10% SDS gels and transferring to PVDF membranes, proteins were probed with antibodies against p53 and glyceraldehyde 3-phosphate dehydrogenase followed by a horseradish peroxidase-conjugated secondary antibody. Detection by the electrochemiluminescence method and capture of immunoblot images were carried out by using FluorChem SP imaging system from Alpha Innotech as previously described .
Polymerase chain reaction.
This work was supported by a grant from Oklahoma Center for the Advancement of Science & Technology (to ZJ Zhao).
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