Mutant RAS selectively promotes sensitivity of myeloid leukemia cells to apoptosis by a protein kinase C-dependent process.

RAS mutations arise at high frequency in human malignancy and have been shown to play a role in the disruption of both normal differentiation and proliferation. In addition, RAS influences a number of intracellular signaling pathways, which impinge on proteins that regulate programmed cell death. In this study, we have examined whether this oncogene can influence the activation of the apoptotic process induced by a range of therapeutic agents used to treat leukemia, and we have identified the downstream targets of RAS mediating the observed changes in sensitivity. Using myeloid leukemia cells (P39) retrovirally transduced with mutant H-RAS, we found that the influence of this oncogene was highly dependent on the inducer used: whereas RAS had no significant effect on spontaneous apoptosis or on the response to the cytotoxic drugs (doxorubicin or 1-beta-arabinofuranosylcytosine), P39-RAS cells showed a strongly augmented response to all-trans-retinoic acid (ATRA) in both the induction of apoptosis and differentiation. Because, under some circumstances, RAF has been associated with promoting apoptosis, we examined whether the activation of this kinase by mutant RAS could be responsible for the augmented response to ATRA. However, constitutive activation of RAF did not alter the apoptotic sensitivity of these cells, making it unlikely that RAS promotes apoptosis by stimulating this kinase. Nor did we find that BCL-2 was differentially down-regulated in P39-RAS cells. Rather, we found that the activation of protein kinase C (PKC) by low-dose phorbol ester could almost entirely recapitulate transformation by RAS, in terms of promoting both apoptosis and differentiation after treatment with ATRA. Moreover, the RAS-induced phenotype could be completely abolished by a specific inhibition of PKC under conditions that had no effect on the response of control cells. In conclusion, we have shown that mutant RAS promotes differentiation-associated cell death in P39 cells by stimulating the activity of PKC, which is itself an important regulator of myeloid differentiation. PKC activation, in turn, powerfully synergizes with the PKC-independent action of ATRA. This work identifies a possible explanation for the ability of this oncogene to promote myeloid differentiation of hematopoietic cells. Clinically, it raises the possibility that although leukemias expressing mutant RAS may not show an altered response to cytotoxic agents, they may show enhanced sensitivity to differentiation therapy with ATRA.

Prevalence of vitamin D inadequacy in Scottish adults with non-vertebral fragility fractures.

BACKGROUND: It is well established that vitamin D levels are sub-optimal in the elderly and that adults with fragility fracture are more likely to have serum vitamin D levels either lower than those of control patients of similar age, or below the normal range. OBJECTIVES: To investigate the prevalence of vitamin D inadequacy in an elderly population presenting to the South Glasgow Fracture Liaison Service with non-vertebral fragility fractures in order to assess the extent of the problem. RESEARCH DESIGN AND METHODS: The retrospective arm of this study used data from an established database to identify patients aged over 50 years admitted to South Glasgow University Hospitals over the previous 4 years with hip fracture. The prospective arm identified the first 50 patients aged over 50 presenting with a clinical non-vertebral fragility fracture with osteoporosis as measured by axial spine and/or hip DEXA (T-score < -2.5) after November 2004. RESULTS: In the retrospective arm, 626 patients were identified from the database: mean age 80.5 years; 94% were aged over 60 and 74% were aged over 75. Data analysis was limited to 548 patients aged over 60 years with vitamin D recordings and not receiving supplementation with calcium and vitamin D. The mean vitamin D level was 24.7 nmol/L (9.9 ng/ml) SD = 17, however, it is likely that the true mean is lower since in approximately 25% of cases vitamin D levels were reported as < 15 nmol/L (effectively unrecordable). These were transcribed as 15 nmol/L in order to permit a numerical value to be calculated. In the absence of an agreement on what should constitute a diagnostic serum level of vitamin D inadequacy, a number of thresholds were considered--97.8% had vitamin D levels below 70 nmol/L and 91.6% had vitamin D levels below 50 nmol/L. There were no significant differences by patient sex, age or season of presentation. The mean age of patients in the prospective arm was 65.8 years (range 50.6-83.8), 72% were aged over 60 and 16% were aged over 75. The mean vitamin D level was 44.1 nmol/L (18.4 ng/ml) SD = 25.3; 82% had vitamin D levels below 70 nmol/L and 72% had vitamin D levels below 50 nmol/L. Although numbers were too small to justify extensive subgroup analyses, the mean vitamin D level in the 13 patients with hip fracture (34.5 nmol/L) was lower than in the 37 with non-hip fractures (48.2 nmol/L). CONCLUSIONS: This study confirms almost universal vitamin D inadequacy among 548 elderly patients admitted to hospital with hip fracture, regardless of whether a threshold of 50 nmol/L or 70 nmol/L was used. However, among a prospective subset of 50 patients with clinical fragility fractures, especially those with non-hip fractures, the prevalence of inadequacy was substantially lower. It may be that vitamin D represents a correctable risk factor for fragility fracture in the elderly, possibly specifically for the hip.

The prevalence of vertebral fracture amongst patients presenting with non-vertebral fractures.

INTRODUCTION: Despite vertebral fracture being a significant risk factor for further fracture, vertebral fractures are often unrecognised. A study was therefore conducted to determine the proportion of patients presenting with a non-vertebral fracture who also have an unrecognised vertebral fracture. METHODS: Prospective study of patients presenting with a non-vertebral fracture in South Glasgow who underwent DXA evaluation with vertebral morphometry (MXA) from DV5/6 to LV4/5. Vertebral deformities (consistent with fracture) were identified by direct visualisation using the Genant semi-quantitative grading scale. RESULTS: Data were available for 337 patients presenting with low trauma non-vertebral fracture; 261 were female. Of all patients, 10.4% were aged 50-64 years, 53.2% were aged 65-74 years and 36.2% were aged 75 years or over. According to WHO definitions, 35.0% of patients had normal lumbar spine BMD (T-score -1 or above), 37.4% were osteopenic (T-score -1.1 to -2.4) and 27.6% osteoporotic (T-score -2.5 or lower). Humerus (n=103, 31%), radius-ulna (n=90, 27%) and hand/foot (n=53, 16%) were the most common fractures. For 72% of patients (n=241) the presenting fracture was the first low trauma fracture to come to clinical attention. The overall prevalence of vertebral deformity established by MXA was 25% (n=83); 45% (n=37) of patients with vertebral deformity had deformities of more than one vertebra. Of the patients with vertebral deformity and readable scans for grading, 72.5% (58/80) had deformities of grade 2 or 3. Patients presenting with hip fracture, or spine T-score