CDKN2A-independent role of BMI1 in promoting growth and survival of Ph+ acute lymphoblastic leukemia.

BMI1 is a key component of the PRC1 (polycomb repressive complex-1) complex required for maintenance of normal and cancer stem cells. Its aberrant expression is detected in chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia (ALL), but no data exist on BMI1 requirement in ALL cells. We show here that BMI1 expression is important for proliferation and survival of Ph+ ALL cells and for leukemogenesis of Ph+ cells in vivo. Levels of BIM, interferon-α (IFNα)-regulated genes and E2F7 were upregulated in BMI1-silenced cells, suggesting that repressing their expression is important for BMI1 biological effects. Consistent with this hypothesis, we found that: (i) downregulation of BIM or E2F7 abrogated apoptosis or rescued, in part, the reduced proliferation and colony formation of BMI1 silenced BV173 cells; (ii) BIM/E2F7 double silencing further enhanced colony formation and in vivo leukemogenesis of BMI1-silenced cells; (iii) overexpression of BIM and E2F7 mimicked the effect of BMI1 silencing in BV173 and SUP-B15 cells; and (iv) treatment with IFNα suppressed proliferation and colony formation of Ph+ ALL cells. These studies indicate that the growth-promoting effects of BMI1 in Ph+ ALL cells depend on suppression of multiple pathways and support the use of IFNα in the therapy of Ph+ ALL.

JAK inhibitors suppress t(8;21) fusion protein-induced leukemia.

Oncogenic mutations in components of the JAK/STAT pathway, including those in cytokine receptors and JAKs, lead to increased activity of downstream signaling and are frequently found in leukemia and other hematological disorders. Thus, small-molecule inhibitors of this pathway have been the focus of targeted therapy in these hematological diseases. We previously showed that t(8;21) fusion protein acute myeloid leukemia (AML)1-ETO and its alternatively spliced variant AML1-ETO9a (AE9a) enhance the JAK/STAT pathway via downregulation of CD45, a negative regulator of this pathway. To investigate the therapeutic potential of targeting JAK/STAT in t(8;21) leukemia, we examined the effects of a JAK2-selective inhibitor TG101209 and a JAK1/2-selective inhibitor INCB18424 on t(8;21) leukemia cells. TG101209 and INCB18424 inhibited proliferation and promoted apoptosis of these cells. Furthermore, TG101209 treatment in AE9a leukemia mice reduced tumor burden and significantly prolonged survival. TG101209 also significantly impaired the leukemia-initiating potential of AE9a leukemia cells in secondary recipient mice. These results demonstrate the potential therapeutic efficacy of JAK inhibitors in treating t(8;21) AML.

p53 stabilization induces apoptosis in chronic myeloid leukemia blast crisis cells.

Philadelphia chromosome positive chronic myeloid leukemia has a progressive course starting in a benign phase and terminating in a blastic phase. In this study, we show that human homolog double minute 2 (HDM2) inhibition, with MI-219-a novel compound, and consequently p53 stabilization induce chronic myeloid leukemia (CML) blast crisis cells to undergo apoptosis regardless of the presence of the T315I mutation in the BCR-ABL kinase domain. The response to MI-219 is associated with the downregulation of c-Myc and the induction of p21(WAF1). The p53 target and pro-apoptotic proteins PUMA, Noxa and Bax are induced, whereas full length Bid protein decreases with increased activity of pro-apoptotic cleaved Bid, and decrease of Mcl-1 is observed by increased caspase activity. CD95/FAS (FAS antigen) receptor is also induced by MI-219, indicating that both intrinsic and extrinsic apoptotic responses are transcriptionally induced. In addition, p53 protein accumulates in the mitochondrial fraction of treated cells involved in transcription-independent induction of apoptosis. We conclude that HDM-2 inhibition with MI-219 effectively induces p53-dependent apoptosis in most blast crisis CML cells, with or without BCR-ABL mutation(s).

An inhibitor-resistant mutant of Hck protects CML cells against the antiproliferative and apoptotic effects of the broad-spectrum Src family kinase inhibitor A-419259.

Chronic myelogenous leukemia (CML) is driven by Bcr-Abl, a constitutively active protein-tyrosine kinase that stimulates proliferation and survival of myeloid progenitors. Global inhibition of myeloid Src family kinase (SFK) activity with the broad-spectrum pyrrolo-pyrimidine inhibitor, A-419259, blocks proliferation and induces apoptosis in CML cell lines, suggesting that transformation by Bcr-Abl requires SFK activity. However, the contribution of Hck and other individual SFKs to Bcr-Abl signaling is less clear. Here, we developed an A-419259-resistant mutant of Hck by replacing the gatekeeper residue (Thr-338; c-Src numbering) in the inhibitor-binding site with a bulkier methionine residue (Hck-T338M). This substitution reduced Hck sensitivity to A-419259 by more than 30-fold without significantly affecting kinase activity in vitro. Expression of Hck-T338M protected K-562 CML cells and Bcr-Abl-transformed TF-1 myeloid cells from the apoptotic and antiproliferative effects of A-419259. These effects correlated with persistence of Hck-T338M kinase activity in the presence of the compound, and were accompanied by sustained Erk and Stat5 activation. In contrast, control cells expressing equivalent levels of wild-type Hck retained sensitivity to the inhibitor. We also show for the first time that A-419259 induces cell-cycle arrest and apoptosis in primary CD34(+) CML cells with equal potency to imatinib. These data suggest that Hck has a nonredundant function as a key downstream signaling partner for Bcr-Abl and may represent a potential drug target in CML.

The multi-functional cellular adhesion molecule CD44 is regulated by the 8;21 chromosomal translocation.

The 8;21 translocation is a common chromosomal abnormality in acute myeloid leukemia (AML). We recently identified a naturally occurring leukemogenic splice variant, AML1-ETO9a (acute myeloid leukemia-1 transcription factor and the eight-twenty-one corepressor-9a), of t(8;21). To understand the leukemic potential of AML1-ETO9a, we performed microarray analysis with the murine multipotential hematopoietic FDCP-mix A4 cell line. We identified changes in expression of various genes including CD44. CD44 is a type I transmembrane protein and functions as the major cellular adhesion molecule for hyaluronic acid, a component of the extracellular matrix. CD44 is expressed in most human cell types and is implicated in myeloid leukemia pathogenesis. We show that the presence of AML1-ETO9a significantly increased the expression of CD44 at both RNA and protein levels. Furthermore, the CD44 promoter is bound by AML1-ETO9a and AML1-ETO at the chromatin level. In addition, in the AML1-ETO9a leukemia mouse model CD44 is regulated in a cell context-dependent manner. Thus, our observations suggest that AML1-ETO and its splice variant AML1-ETO9a are able to regulate the expression of the CD44 gene, linking the 8;21 translocation to the regulation of a cell adhesion molecule that is involved in the growth and maintenance of the AML blast/stem cells.

Estrogen increases hyperemic microvascular blood flow velocity in postmenopausal women.

BACKGROUND: Epidemiologic studies suggest that estrogen replacement therapy (ERT) is protective against vascular disease. ERT confers this benefit by lowering lipid levels and improving arterial function. However, its effect on the microvasculature in vivo is unknown. Thus the purposes of this study were to evaluate effect of estrogen status on the hyperemic response of the microvasculature in vivo in postmenopausal women and to compare the hyperemic response of the microvasculature in postmenopausal women taking ERT with that of premenopausal women. METHODS: We measured forearm microvasculature flow velocity by using a laser Doppler in a cross section of 64 healthy premenopausal and postmenopausal women 23 to 72 years old. Microvasculature blood flow velocity was measured at baseline. throughout 2 minutes of ischemia, and immediately after the ischemic period was terminated (i.e., during the peak hyperemic response). RESULTS: The peak of the hyperemic flow velocity (PHFV) in the postmenopausal women who were taking long-term ERT at usual doses was greater than that of postmenopausal women who were not currently taking ERT (p < .0001). Moreover, the PHFV of postmenopausal women taking ERT was similar to that of premenopausal women. Multivariate regression analysis showed estrogen status and baseline flow velocity to be independent predictors of PHFV. CONCLUSIONS: Current, long-term ERT at usual replacement doses is associated with improved microvascular responses in postmenopausal women, which may explain some of its beneficial vascular effects.

Exclusion of the nuclear factor-kappa B3 (REL A) gene as candidate for the multiple endocrine neoplasia type 1 (MEN 1) gene.

Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by neoplasia and hyperplasia in specific endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a region of approximately 900 kb on chromosome 11q13. The nuclear factor-kappa B (NF-kappa B) is a transcription factor with pleiotropic expression, which is involved in the regulation of expression of many cellular genes. The p50/p65 heterodimer is the most abundant form of NF-kappa B. The gene encoding the p65 subunit (NF-kappa B3/REL A) was recently localized in the 900-kb MEN 1 region and was considered a good candidate gene for MEN 1. The structure and nucleotide sequence of the NF-kappa B3 coding region in MEN 1 patients were compared with those of non-MEN 1 subjects, to determine the potential role of this gene in MEN 1 tumorigenesis. Southern blot analysis with constitutional DNA from probands of 14 independent MEN 1 families and DNA from four MEN 1 tumor specimens did not reveal any structural abnormality of the NF-kappa B3 gene. Direct sequencing of cDNAs from two affected subjects from 2 different MEN 1 families, as well as nucleotide sequence analysis of exon/intron boundaries in these patients, did not reveal MEN 1-specific point mutations or other small structural aberrations in the NF-kappa B3 gene. These results make it very unlikely that the NF-kappa B3 gene is the gene responsible for the development of MEN 1.

Revision total knee arthroplasty.

Careful attention to axial alignment, soft tissue balance, and stability will minimize prosthetic failure. In revision arthroplasty a prosthesis designed to replace bone loss with the least constraint possible should be used. In the current series revision of the noninfected failed total knee arthroplasty has provided satisfactory results in 50% to 60% of the patients. We believe that use of the newer implants and instrumentation will improve results markedly.

Polycentric total knee arthroplasty. A ten-year follow-up study.

After review of the first 209 polycentric total knee arthroplasties (in 159 patients) performed at the Mayo Clinic between July 1970 and November 1971, we found that the calculated probability of the arthroplasty remaining successful ten years postoperatively was 66 per cent. Actual results showed 42 per cent of the arthroplasties to be successful in patients who were still alive at review; another 24 per cent were successful but were in patients who had died or were lost to follow-up before ten years postoperatively. In 34 per cent failure occurred, which we defined as reoperation for any reason, unacceptable pain, or loss of function. The most common causes of failure were instability or ligament laxity (13 per cent), loosening of a component (7 per cent), infection (3 per cent), and patellofemoral joint pain (4 per cent). Prior knee surgery significantly decreased the probability of success, as did axial malalignment of the prosthetic components at operation.

Unicompartmental knee arthroplasty using polycentric and geometric hemicomponents.

Unicompartmental knee arthroplasty was performed in 207 knees of 179 patients using either a polycentric (188) or a geometric (nineteen) hemicomponent. After an average follow-up of 2.6 years, the results in 184 (89 per cent) of the knees were satisfactory. Pain and the need for ambulatory aids were reduced, and the distance the patients could walk was increased. Twenty-three (11 per cent) of the 207 procedures were rated as failures. The major cause of failure was loosening of the components (tibial in twelve and femoral in one); there also were unexplained pain in five knees, problems with the opposite unreplaced compartment in three, technical error in one, and pain in the patellofemoral joint in one. Nine of 155 intraoperative specimens for bacterial culture obtained during unicompartmental arthroplasty were positive, and two specimens that were positive on culture were obtained during revision of twenty failed unicompartmental arthroplasties. No gross or histological evidence of infection was demonstrated at operation. Based on this study, we concluded that this procedure can provide satisfactory relief of pain, adequate knee motion, and increased levels of independence and activity for patients with unicompartmental disease who are not suitable candidates for proximal tibial osteotomy or total knee replacement.

Total joint arthroplasty. The knee.

Total knee replacement has become an established form of treatment for gonarthrosis and usually results in excellent relief of pain and approximately 90 degrees of joint motion with satisfactory joint stability. The anatomic stability that cannot be restored at surgery must be provided for by additional prosthetic stability. Fixation of prosthetic devices, particularly in the tibia, is marginal and results in an increased incidence of loosening when the quality of bone is weak, as in osteoporosis, or when shear stress is increased because of malalignment or prosthetic constraint. Resurfacing techniques provide the greatest options if surgical revision is necessary. Surgical goals should be realistically assessed so as to maintain the best potential for future treatment options.

Arthrodesis of the knee following failed total knee arthroplasty.

In forty-five patients, who had an arthrodesis because of failed total knee arthroplasty, the cause was infection in forty, instability in two, failure of the prosthesis in two, and loosening in one. The arthrodesis succeeded in twenty-nine (81%) of thirty-six patients who had had a minimally or partially constrained arthroplasty and in five (56%) of nine who had had a hinge-type prosthesis inserted. The reasons for failure were severe bone loss, persistent sepsis, and loss of bone apposition after manipulation. The technique of arthrodesis did not seem to influence the final result. External fixation most commonly had to be used because of the infections and the device was kept in place for an average of ten weeks, after which immobilization in a cast was used until the arthrodesis healed.

Total hip arthroplasty: an editiorial comment.

Total hip arthroplasty has become an accepted method of management of severe painful problems of the hip. It has undergone some dramatic changes, the major thrust now being to more nearly match the mechanical characteristics of the implant to the bone and cartilage they replace.

Current status of total knee arthroplasty.

Total knee arthroplasty has become an acceptable method of surgical management for severe, disabling gonarthropathy. The three major biomechanical classifications of total knee prostheses are minimally constrained, partially constrained, and fully constrained. The major indication for total knew arthroplasty is pain, followed in a much lower frequency by instability, loss of motion, and deformity. The principal contraindications for the various types relate solely to the residual or restorable ligamentous stability of the knee and the degree of bone loss. In general, the greater the instability and bone loss, the more constrained the prosthesis must be Theoretically, loosening rates increase with increasing shear stresses, which are generally highest with the most constrained prostheses. The major complications are sepsis, loosening, and instability. Various prostheses have incorporated patellofemoral resurfacing as the final dimension in producing a total knee arthroplasty. Knee arthroplasty is very effective in preserving functional knee motion, with relief of pain as an alternative to arthrodesis.

Polycentric total knee arthroplasty. A two-year follow-up study.

Polycentric total knee arthroplasty provided significant relief of pain in 86 per cent of 500 knees. The independence and activity levels of the patients increased dramatically. The frequency of major complications as reflected by reoperation was 10 per cent in this series. There was a 2.8 per cent deep infection rate. One-third of the infected knees were salvaged and two-thirds required arthrodesis. Loosening of a component was noted in 2.4 per cent. After operation the average range of motion was from 6 to 101 degrees of flexion, for a range of 95 degrees; this was a 5-degree increase over average preoperative motion. Ninety-six per cent of the patients expressed satisfaction with the surgical result.

A statistical evaluation of polycentric total knee arthroplasties.

Four hundred nineteen knees in 299 patients were examined preoperatively and at two years after polycentric total knee surgery. One hundred forty-three knees were in 109 patients with osteoarthritis. Two hundred seventy-six knees were in 190 patients with rheumatoid arthritis. Pain decreased significantly, use of aids decreased significantly, and distance walked increased significantly in both groups. Rheumatoid patients did not fare as well as osteoarthritics in the use of aids and distance walked. Osteoarthritic knees had proportionately more implant settling, implant loosening and reoperations than did rheumatoid arthritic knees.