The Role Of Dickkopf1 In Multiple Myeloma

Part I:Clinical study of DKK1 in multiple myelomaObjective To detect bone marrow plasma and serum concentrations of Dickkopf1(DKK1) in patients with multiple myeloma and perform a correlative analysis between DKK1, myeloma stage and lytic bone disease and evaluate the effect of different treatment regimens for MM on serum DKK1 and the relationship with the response to treatment.Methods DKK1 bone marrow plasma and serum levels were quantified in 80 newly diagnosed MM patients,10 monoclonal gammopathy of undetermined significance (MGUS) patients and 20 control subjects by ELISA, using a monoclonal anti-DKK1 antibody. And serum samples were collected from 136 patients with MM treated with bortezomib and dexamethasone (n = 72), thalidomide and dexamethasone (n = 64) before and 3 months after initiation of therapy.Results Bone marrow plasma DKK1 was elevated in MM as compared with MGUS (mean 9.3588ng/ml vs. 1.762ng/ml; P=0.001). There was no difference between MGUS patients and control subjects(1.762ng/ml vs 1.2491ng/ml; P > 0.05). Bone marrow plasma DKK1 levels significantly correlated with myeloma stage according to Durie and Salmon (mean 2.4410ng/ml vs. 10.3471ng/ml in stage I and II ? III, respectively; P = 0.002). There was no difference between MM patients in stage I and MGUS patients(P>0.05). There were similar results using ISS stage. Importantly, myeloma patients without lytic lesions in conventional radiography had significantly lower DKK1 levels than patients with lytic bone disease (mean 2.4348 ng/ml vs. 11.2272ng/ml; P<0.001). Of interest, bone marrow plasma DKK1 correlated with the number of bone lesions (0 vs. 1–3 vs. >3 lesions: 2.4348ng/ml vs. 4.9845ng/ml vs. 15.3342ng/ml; P<0.001). In this study, the effect of different treatment regimens for MM patients on serum DKK1 was evaluated and correlated with the response to treatment in 136 myeloma patients receiving bortezomib and dexamethasone(n=72), thalidomide and dexamethasone(n=64). A significant decrease of DKK1 after therapy was seen in the Bortezomib and dexamethasone groups (mean 14.1789ng/ml vs. 8.3972ng/ml; P < 0.05). Thalidomide and dexamethasone led to a nonsignificant decrease in DKK1 (10.5614ng/ml vs. 9.5326ng/ml; P>0.05). Within all groups, a significant decrease of DKK1 was only seen in responders (i.e. patients achieving complete remission or partial remission), but not in non-responders.Conclusion Myeloma patients have increased bone marrow plasma DKK1 as compared with patients with MGUS and control subjects. Bone marrow plasma concentrations of DKK1 have close relationship with myeloma stage and lytic bone disease. Serum DKK1 levels decrease in myeloma patients responding to treatment, corelate with the regimen chosen.Part II: Gene expression of DKK1 receptor LRP5/6 and Kremen1/2 in myeloma cells and stromal cellsObjective To compare the gene expression of LRP5/6,Kremen1/2 andβ-catenin in Human myeloma cell lines, CD138+ primary myeloma cells, stromal cells of multiple myeloma patients and healthy volunteers and investigate the effect of multiple myeloma cells or rhDKK1on gene expression in stromal cells of healthy volunteers and identify the role of DKK1 in multiple myeloma.Methods DKK1 protein expression was checked in human myeloma cell lines, CD138+ primary myeloma cells, stromal cells and myeloma cell condition media using Western Blot analysis and ELISA assay. LRP5/6,Kremen1/2 andβ-catenin mRNA expression in Human myeloma cell lines, CD138+ primary myeloma cells, stromal cells of multiple myeloma patients and healthy volunteers were tested by Realtime-PCR. To compare the effect of multiple myeloma cells or rhDKK1 on gene expression in human stromal cells, we did Real-time PCR for LRP5/6,Kremen1/2 andβ-catenin mRNA expression in stromal cells of healthy volunteers cocultured with multiple myeloma cells or rhDKK1.Results DKK1 was produced by multiple myeloma cells, not stromal cells. DKK1 was detectable in myeloma cell condition media, as well. LRP5/6 and Kremen1/2 mRNA expression in stromal cells of multiple myeloma patients were higher than that in stromal cells of healthy volunteer and CD138+ primary myeloma cells.β-catenin mRNA expression in CD138+ primary myeloma cells was higher than that in stromal cells of multiple myeloma patients and healthy volunteers. Gene expression of LRP5/6, Kremen1/2 was elevated in stromal cells of healthy volunteers after 48h -72h of coculture with HMCLs RPMI-8226 in the presence or absence of a Transwell insert. A significant inhibitory effect was observed on the expression ofβ-catenin in stromal cells of healthy volunteers after 48h -72h of coculture with multiple myeloma cells. No significant effect was observed on the gene expression after 24h coculture. Similar results were obtained with the HMCLs NCI-H929 in coculture (data not shown). We examined the effect of rhDKK1 on LRP5/6, Kremen1/2 andβ-catenin mRNA expression in stromal cells of healthy volunteers. The levels of LRP5/6 and Kremen1/2 gene expression in stromal cells of healthy volunteers were highest at 72h post rhDKK1(at concentration 700 ng/mL) treatment. Andβ-catenin mRNA expression in stromal cells of healthy volunteers decreased significantly at the same time.Conclusion LRP5/6,Kremen1/2 andβ-catenin gene were expressed differently in human myeloma cell lines, CD138+ primary myeloma cells and stromal cells. And DKK1 produced by myloma cells may play an important role in the different gene expression in stromal cells between multiple myeloma patients and healthy volunteers.Part III: Expression,purification and functional examination of DKK1Objective To express, purify DKK1, and investigate the binding capacity of its receptor on the cell membrane of myeloma cells and stromal cells.Methods Vector pcDNA3.1 (-)-DKK1 was transfected into 293T cells with LipofectAMINETM2000 reagent. DKK1protein in the supernatant was identified by western blot. With the Ni-Agrose His protein purification kit, DKK1 protein was purified. Flow cytometry (FCM) was used to study DKK1-bound receptor on the cell membrane of myeloma cells and stromal cells.Results With LipofectAMINETM2000 reagent, the mammalian expression vector pcDNA3.1 (-)-DKK1 was successfully transfected into 293T cells. In the supernatant, DKK1 protein was collected. Using anti-DKK1 antibody and anti-His antibody, DKK1 fusion protein in the supernatant was identified by western blot. Flow cytometry results showed that DKK1 protein could specifically bind the receptor on the cell membrane of myeloma cells and stromal cells. The mean fluorescence intensity (MFI) of receptor on myeloma cells, stromal cells of the healthy volunteers and myeloma patients were (58.6±17.2), (20.8±8.4), (104.5±32.1), respectively. There were significantly difference among three groups(P<0.05).Conclusion Using mammalian expression vector, DKK1 protein was obtained and showed a high biological activity and could specifically bind the receptor. The amount of receptor of DKK1 protein specifically binding on the cell membrane of myeloma cells and stromal cells were different.