First-in-human autologous implantation of genetically modified adipocytes expressing LCAT for the treatment of familial LCAT deficiency.

Background: Familial lecithin: cholesterol acyltransferase (LCAT) deficiency (FLD) is a severe inherited disease without effective treatment. Patients with FLD develop severe low HDL, corneal opacity, hemolytic anemia, and renal injury. Objective: We developed genetically modified adipocytes (GMAC) secreting LCAT (LCAT-GMAC) for ex vivo gene therapy. GMACs were prepared from the patient's adipocytes to express LCAT by retroviral gene transduction to secrete functional enzymes. This study aimed to evaluate the safety and efficacy of LCAT-GMAC implantation in an FLD patient. Methods: Proliferative preadipocytes were obtained from a patient using a ceiling culture and retrovirally transduced with LCAT. After obtaining enough cells by expansion culture of the transduced cells, the resulting LCAT-GMACs were implanted into a patient with FLD. To evaluate the safety and efficacy, we analyzed the outcome of the autologous implantation for 24 weeks of observation and subsequent 240 weeks of the follow-up periods. Results: This first-in-human autologous implantation of LCAT-GMACs was shown to be safe by evaluating adverse events. The LCAT-GMAC implantation increased serum LCAT activity by approximately 50% of the baseline and sustained over three years. Consistent with increased LCAT activity, intermediate-density lipoprotein (IDL) and free cholesterol levels of the small and very small HDL fractions decreased. We found the hemoglobin/haptoglobin complex in the hemolyzed pre-implantation sera of the patient. After one week of the implantation, the hemoglobin/haptoglobin complex almost disappeared. Immediately after the implantation, the patient's proteinuria decreased temporarily to mild levels and gradually increased to the baseline. At 48 weeks after implantation, the patient's proteinuria deteriorated with the development of mild hypertension. By the treatment with antihypertensives, the patient's blood pressure normalized. With the normalization of blood pressure, the proteinuria rapidly decreased to mild proteinuria levels. Conclusions: LCAT-GMAC implantation in a patient with FLD is shown to be safe and appears to be effective, in part, for treating anemia and proteinuria in FLD.

A novel homozygous frameshift mutation in the APOA1 gene associated with marked high-density lipoprotein deficiency.

The proband was a 53-year-old Japanese woman. Despite having no atherosclerotic vascular lesions on a physiological examination, markedly decreased levels of high-density lipoprotein (HDL) were always noted at her annual medical checkup. She also had corneal opacities but neither xanthoma nor tonsillar hypertrophy. A biochemical examination showed decreased levels of both apolipoprotein A-I (apoA-I) (<5 mg/dL) and lecithin-cholesterol acyltransferase (LCAT) activity. Her brother and son also had low concentrations of HDL-cholesterol, suggesting the presence of a genetic abnormality. Therefore, a sequence analysis of the genes for ABCA1, LCAT and apoA-I proteins was performed in the proband. The analysis of the APOA1 gene revealed a novel homozygous two-nucleotide deletion in exon 4 (c.614_615delTC), which causes a frameshift after residue 205 of the apoA-I protein (p.Leu205fs). Since no mutation has been found in the ABCA1 or LCAT gene, functional abnormalities of the carboxyl-terminal region of the apoA-I protein in lipid binding might have caused the low HDL-cholesterol levels and decreased LCAT activity, possibly associated with corneal opacities but not premature CAD, in the patient.

Lipoprotein subfractions highly associated with renal damage in familial lecithin:cholesterol acyltransferase deficiency.

OBJECTIVE: In familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD), deposition of abnormal lipoproteins in the renal stroma ultimately leads to renal failure. However, fish-eye disease (FED) does not lead to renal damage although the causative mutations for both FLD and FED lie within the same LCAT gene. This study was performed to identify the lipoproteins important for the development of renal failure in genetically diagnosed FLD in comparison with FED, using high-performance liquid chromatography with a gel filtration column. APPROACH AND RESULTS: Lipoprotein profiles of 9 patients with LCAT deficiency were examined. Four lipoprotein fractions specific to both FLD and FED were identified: (1) large lipoproteins (>80 nm), (2) lipoproteins corresponding to large low-density lipoprotein (LDL), (3) lipoproteins corresponding to small LDL to large high-density lipoprotein, and (4) to small high-density lipoprotein. Contents of cholesteryl ester and triglyceride of the large LDL in FLD (below detection limit and 45.8±3.8%) and FED (20.7±6.4% and 28.0±6.5%) were significantly different, respectively. On in vitro incubation with recombinant LCAT, content of cholesteryl ester in the large LDL in FLD, but not in FED, was significantly increased (to 4.2±1.4%), whereas dysfunctional high-density lipoprotein was diminished in both FLD and FED. CONCLUSIONS: Our novel analytic approach using high-performance liquid chromatography with a gel filtration column identified large LDL and high-density lipoprotein with a composition specific to FLD, but not to FED. The abnormal lipoproteins were sensitive to treatment with recombinant LCAT and thus may play a causal role in the renal pathology of FLD.

Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system.

Auto-transplantation of adipose tissue is commonly used for the treatment of tissue defects in plastic surgery. The survival of the transplanted adipose tissue is not always constant, and one of reasons is the accelerated apoptosis of the implanted preadipocytes. We have recently established highly homogeneous preadipocytes, named ccdPAs. The aim of the current study was to evaluate the regulation of the potency of platelet-rich plasma (PRP) on the apoptosis of ccdPAs in vitro. PRP stimulated the proliferation of the preadipocytes in a dose-dependent manner, and the stimulatory activity of 2% PRP was significantly higher than that of 2% FBS or 2% platelet-poor plasma (PPP). The presence of 2% PRP significantly inhibited serum starvation- or TNF-α/cycloheximide-induced apoptosis in comparison to 2% FBS or 2% PPP. DAPK1 and Bcl-2-interacting mediator of cell death (BIM) mRNAs were reduced in the preadipocytes cultured with 2% PRP in comparison to those cultured in 2% FBS. The gene expression levels were significantly higher in cells cultured without serum in comparison to cells cultured with 2% FBS, and the levels in the cells with 2% PRP were reduced to 5-10% of those in the cells without serum. These results indicated that ccdPAs exhibit anti- apoptotic activities, in addition to increased proliferation, when cultured in 2% PRP in comparison to the same concentration of FBS, and that this was accompanied with reduced levels of DAPK1 and BIM mRNA expression in in vitro culture. PRP may improve the outcome of transplantation of adipose tissue by enhancing the anti-apoptotic activities of the implanted preadipocytes.

Fibrin glue is a candidate scaffold for long-term therapeutic protein expression in spontaneously differentiated adipocytes in vitro.

Adipose tissue is expected to provide a source of cells for protein replacement therapies via auto-transplantation. However, the conditioning of the environment surrounding the transplanted adipocytes for their long-term survival and protein secretion properties has not been established. We have recently developed a preparation procedure for preadipocytes, ceiling culture-derived proliferative adipocytes (ccdPAs), as a therapeutic gene vehicle suitable for stable gene product secretion. We herein report the results of our evaluation of using fibrin glue as a scaffold for the transplanted ccdPAs for the expression of a transduced gene in a three-dimensional culture system. The ccdPAs secreted the functional protein translated from an exogenously transduced gene, as well as physiological adipocyte proteins, and the long viability of ccdPAs (up to 84 days) was dependent on the fibrinogen concentrations. The ccdPAs spontaneously accumulated lipid droplets, and their expression levels of the transduced exogenous gene with its product were maintained for at least 56 days. The fibrinogen concentration modified the adipogenic differentiation of ccdPAs and their exogenous gene expression levels, and the levels of exogenously transduced gene expression at the different fibrinogen concentrations were dependent on the extent of adipogenic differentiation in the gel. These results indicate that fibrin glue helps to maintain the high adipogenic potential of cultured adipocytes after passaging in a 3D culture system, and suggests that once they are successfully implanted at the transplantation site, the cells exhibit increased expression of the transduced gene with adipogenic differentiation.

Ceiling culture-derived proliferative adipocytes retain high adipogenic potential suitable for use as a vehicle for gene transduction therapy.

Adipose tissue is expected to provide a source of proliferative cells for regenerative medicine and cell-transplantation therapies using gene transfer manipulation. We have recently identified ceiling culture-derived proliferative adipocytes (ccdPAs) from the mature adipocyte fraction as cells suitable as a therapeutic gene vehicle because of their stable proliferative capacity. In this study, we examined the capability of adipogenic differentiation of the ccdPAs compared with stromal vascular fraction (SVF)-derived progenitor cells (adipose-derived stem cells, ASCs) with regard to their multipotential ability to be converted to another lineage and therefore their potential to be used for regenerative medicine research. After in vitro passaging, the surface antigen profile and the basal levels of adipogenic marker genes of the ccdPAs were not obviously different from those of the ASCs. However, the ccdPAs showed increased lipid-droplet accumulation accompanied with higher adipogenic marker gene expression after stimulation of differentiation compared with the ASCs. The higher adipogenic potential of the ccdPAs than the ASCs from the SVF was maintained for 42 days in culture. Furthermore, the difference in the adipogenic response was enhanced after partial stimulation without indomethacin. These results indicate that the ccdPAs retain a high adipogenic potential even after in vitro passaging, thus suggesting the commitment of ccdPAs to stable mature adipocytes after autotransplantation, indicating that they may have potential for use in regenerative and gene-manipulated medicine.

Fibrin glue increases the cell survival and the transduced gene product secretion of the ceiling culture-derived adipocytes transplanted in mice.

The development of clinically applicable scaffolds is important for the application of cell transplantation in various human diseases. The aims of this study are to evaluate fibrin glue in a novel protein replacement therapy using proliferative adipocytes and to develop a mouse model system to monitor the delivery of the transgene product into the blood and the fate of the transduced cells after transplantation. Proliferative adipocytes from mouse adipose tissue were transduced by a retroviral vector harboring the human lecithin-cholesterol acyltransferase (lcat) gene, and were subcutaneously transplanted into mice combined with fibrin glue. The lcat gene transduction efficiency and the subsequent secretion of the product in mouse adipocytes were enhanced using a protamine concentration of 500 µg/ml. Adipogenesis induction did not significantly affect the lcat gene-transduced cell survival after transplantation. Immunohistochemistry showed the ectopic enzyme production to persist for 28 days in the subcutaneously transplanted gene- transduced adipocytes. The increased viability of transplanted cells with fibrin glue was accompanied with the decrease in apoptotic cell death. The immunodetectable serum LCAT levels in mice implanted with the fibrin glue were comparable with those observed in mice implanted with Matrigel, indicating that the transplanted lcat gene-transduced adipocytes survived and functioned in the transplanted spaces with fibrin glue as well as with Matrigel for 28 days. Thus, this in vivo system using fibrin is expected to serve as a good model to further improve the transplanted cell/scaffold conditions for the stable and durable cell-based replacement of defective proteins in patients with LCAT deficiency.

Disturbed apolipoprotein A-I-containing lipoproteins in fish-eye disease are improved by the lecithin:cholesterol acyltransferase produced by gene-transduced adipocytes in vitro.

We report the in vitro efficacy of recombinant LCAT produced by lcat gene-transduced proliferative adipocytes (ccdPA/lcat), which has been developed for enzyme replacement therapy. ApoA-I-specific immunodetection in combination with 1D and 2D gel electrophoreses showed that the disturbed high-density lipoprotein subpopulation profile was clearly ameliorated by the in vitro incubation with ccdPA/lcat-derived recombinant LCAT. Thus, these results using ccdPA/lcat strongly suggest the cell implantation could contribute the enzyme replacement for the patients with LCAT deficiency.

Simultaneous suppression of MITF and BRAF V600E enhanced inhibition of melanoma cell proliferation.

Microphthalmia-associated transcription factor (MITF) is a master gene regulating differentiation of melanocytes, and a lineage survival oncogene mediating pro-proliferative function in malignant melanoma. However, high expression of MITF also has an anti-proliferative effect. To clarify the therapeutic implication of MITF as a molecular target for human melanoma, we evaluated the role of MITF in cell proliferation in a panel of human melanoma cell lines which express different levels of MITF. We found that both MITF depletion and forced expression of MITF significantly inhibited proliferation, suggesting that endogenous MITF is regulated at an appropriate level for melanoma cell proliferation, and could be a molecular target for melanoma. However, half of the melanoma cell lines in this study were relatively resistant to MITF depletion, indicating other treatment strategies are required for therapy. Our microarray analysis indicated that regulation of several cell growth-associated molecules may be independent of MITF and dependent on BRAF(V600E). Thus to enhance the anti-proliferative effect of MITF down-regulation, we combined shRNA-mediated MITF depletion with BRAF(V600E) inactivation, another known molecular target for melanoma. Indeed, simultaneous depletion of both MITF and BRAF(V600E) significantly inhibited melanoma growth even for the melanoma cell lines resistant to MITF depletion. These results suggest MITF may be an important molecular target for human melanoma and simultaneous inhibition of MITF and MAPK signaling may be an attractive strategy for melanoma treatment.

Estrogen-mediated post transcriptional down-regulation of breast cancer resistance protein/ABCG2.

Breast cancer resistance protein (BCRP)/ABCG2 mediates concurrent resistance to chemotherapeutic agents, such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan, by pumping them out of cells. We previously reported that BCRP transports sulfated estrogens. In the present study, we show that at physiologic levels, estrogens markedly decrease endogenous BCRP expression in the estrogen-responsive and estrogen receptor alpha (ERalpha)-positive human breast cancer MCF-7 cells, but not in estrogen-nonresponsive human cancer cells. 17 beta-Estradiol (E(2)) also significantly reduces exogenous BCRP expression, driven by a constitutive promoter, in BCRP-transduced estrogen-responsive and ERalpha-positive MCF-7 (MCF-7/BCRP) and T-47D cells, but not in BCRP-transduced estrogen-nonresponsive MDA-MB-231 and SKOV-3 cells. E(2) potentiates the cytotoxicity of SN-38, but not vincristine, in MCF-7/BCRP cells significantly, and increases cellular topotecan uptake in MCF-7 and MCF-7/BCRP cells. Antiestrogen tamoxifen partially reverses E(2)-mediated BCRP down-regulation in MCF-7 and MCF-7/BCRP cells and treatment of MCF-7/BCRP cells with an ERalpha small interfering RNA abolished E(2)-mediated BCRP down-regulation, suggesting that interaction of E(2) and ERalpha is necessary for BCRP down-regulation. E(2) does not affect endogenous BCRP mRNA levels in MCF-7 cells or exogenous BCRP mRNA levels in MCF-7/BCRP cells. The results from pulse-chase labeling experiments with MCF-7/BCRP cells suggest that decreased protein biosynthesis and maturation, but not alterations in protein turnover, might underlie E(2)-mediated BCRP down-regulation. These data indicate that estrogen down-regulates BCRP expression by novel posttranscriptional mechanisms. This is the first report of small molecules that can affect BCRP protein expression in cells and may therefore assist in establishing new strategies for regulating BCRP expression.

Gefitinib reverses breast cancer resistance protein-mediated drug resistance.

Breast cancer resistance protein (BCRP) is an ATP binding cassette transporter that confers resistance to a series of anticancer agents such as 7-ethyl-10-hydroxycamptothecin (SN-38), topotecan, and mitoxantrone. In this study, we evaluated the possible interaction of gefitinib, a selective epidermal growth factor receptor tyrosine kinase inhibitor, with BCRP. BCRP-transduced human epidermoid carcinoma A431 (A431/BCRP) cells acquired cellular resistance to gefitinib, suggesting that BCRP could be one of the determinants of gefitinib sensitivity in a certain sort of cells. Next, the effect of gefitinib on BCRP-mediated drug resistance was examined. Gefitinib reversed SN-38 resistance in BCRP-transduced human myelogenous leukemia K562 (K562/BCRP) or BCRP-transduced murine lymphocytic leukemia P388 (P388/BCRP) cells but not in these parental cells. In addition, gefitinib sensitized human colon cancer HT-29 cells, which endogenously express BCRP, to SN-38. Gefitinib increased intracellular accumulation of topotecan in K562/BCRP cells and suppressed ATP-dependent transport of estrone 3-sulfate, a substrate of BCRP, in membrane vesicles from K562/BCRP cells. These results suggest that gefitinib may overcome BCRP-mediated drug resistance by inhibiting the pump function of BCRP. Furthermore, P388/BCRP-transplanted mice treated with combination of irinotecan and gefitinib survived significantly longer than those treated with irinotecan alone or gefitinib alone. In conclusion, gefitinib is shown to interact with BCRP. BCRP expression in a certain sort of cells is supposed to be one of the determinants of gefitinib sensitivity. Gefitinib inhibits the transporter function of BCRP and reverses BCRP-mediated drug resistance both in vitro and in vivo.

Phytoestrogens/flavonoids reverse breast cancer resistance protein/ABCG2-mediated multidrug resistance.

Breast cancer resistance protein (BCRP), also called ABCG2, confers resistance to anticancer agents such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan. We found previously that sulfated estrogens are physiologic substrates of BCRP. Flavonoids with weak estrogenic activities are called phytoestrogens. In this study, we show that phytoestrogens/flavonoids, such as genistein, naringenin, acacetin, and kaempferol, potentiated the cytotoxicity of SN-38 and mitoxantrone in BCRP-transduced K562 (K562/BCRP) cells. Some glycosylated flavonoids, such as naringenin-7-glucoside, also effectively inhibited BCRP. These flavonoids showed marginal effect on the drug sensitivity of K562 cells. Genistein and naringenin reversed neither P-glycoprotein-mediated vincristine resistance nor multidrug resistance-related protein 1-mediated VP-16 resistance. Genistein and naringenin increased cellular accumulation of topotecan in K562/BCRP cells. K562/BCRP cells also accumulated less [(3)H]genistein than K562 cells. [(3)H]genistein transport in the basal-to-apical direction was greater in BCRP-transduced LLC-PK1 (LLC/BCRP) cells, which express exogenous BCRP in the apical membrane, than in parental cells. Fumitremorgin C abolished the increased transport of [(3)H]genistein in LLC/BCRP cells compared with parental cells. TLC analysis revealed that genistein was transported in its native form but not in its metabolized form. These results suggest that genistein is among the natural substrates of BCRP and competitively inhibits BCRP-mediated drug efflux. The results have two important clinical implications: (a) flavonoids and glycosylated flavonoids may be useful in overcoming BCRP-mediated drug resistance in tumor cells; and (b) coadministration of flavonoids with BCRP-substrate antitumor agents may alter the pharmacokinetics and consequently increase the toxicity of specific antitumor agents in cancer patients.

Single amino acid substitutions in the transmembrane domains of breast cancer resistance protein (BCRP) alter cross resistance patterns in transfectants.

Breast cancer resistance protein (BCRP) is a member of ATP-binding cassette transporters that has an N-terminal ATP binding domain and a C-terminal transmembrane domain (TM). Expression of wild-type BCRP confers resistance to multiple chemotherapeutic agents such as mitoxantrone, SN-38 and topotecan, but not to doxorubicin. We made 32 BCRP mutants with an amino acid substitution in the TMs (7 E446-mutants in TM2, 15 R482-mutants in TM3, 4 N557-mutants in TM5 and 6 H630-mutants in TM6) and examined the effect of the substitutions on cellular drug resistance. PA317 cells transfected with any one of the 7 E446-mutant BCRP cDNAs did not show drug resistance. Cells transfected with any one of the 13 R482X2-BCRP cDNAs (X2 = N, C, M, S, T, V, A, G, E, W, D, Q and H, but not Y and K) showed higher resistance to mitoxantrone and doxorubicin than the wild-type BCRP-transfected cells. Cells transfected with N557D-BCRP cDNA showed similar resistance to mitoxantrone but lower resistance to SN-38 than the wild-type BCRP-transfected cells. Cells transfected with N557E-, H630E- or H630L-BCRP cDNA showed similar degrees of resistance to mitoxantrone and SN-38. Estrone and fumitremorgin C reversed the drug resistance of cells transfected with R482-, N557- or H630-mutant BCRP cDNA. Cells transfected with R482G- or R482S-BCRP cDNA showed less intracellular accumulation of [3H]mitoxantrone than the wild-type BCRP-transfected cells. These results suggest that E446 in TM2, R482 in TM3, N557 in TM5 and H630 in TM6 play important roles in drug recognition of BCRP.

Breast cancer resistance protein exports sulfated estrogens but not free estrogens.

Breast cancer resistance protein (BCRP), an ATP-binding cassette transporter, confers resistance to a series of anticancer reagents such as mitoxantrone, 7-ethyl-10-hydroxycamptothecin, and topotecan. We reported previously that estrone and 17beta-estradiol reverse BCRP-mediated multidrug resistance. In the present study, we demonstrate that BCRP exports estrogen metabolites. First, we generated BCRP-transduced LLC-PK1 (LLC/BCRP) cells, in which exogenous BCRP is expressed in the apical membrane, and investigated transcellular transport of 3H-labeled compounds using cells plated on microporous filter membranes. The basal-to-apical transport (excretion) of mitoxantrone, estrone, and 17beta-estradiol was greater in LLC/BCRP cells than in LLC-PK1 cells. Thin-layer chromatography of transported steroids revealed that the transport of estrone and 17beta-estradiol was independent of BCRP expression. Alternatively, increased excretion of estrone sulfate and 17beta-estradiol sulfate was observed in LLC/BCRP cells. BCRP inhibitors completely inhibited the increased excretion of sulfated estrogens across the apical membrane. Conversion of estrogens into their sulfate conjugates was similar between LLC/BCRP and LLC-PK1 cells, suggesting that the increased excretion of estrogen sulfates was attributable to BCRP-mediated transport. Next, the uptake of 3H-labeled compounds in membrane vesicles from BCRP-transduced K562 (K562/BCRP) cells was investigated. 3H-labeled estrone sulfate, but not 3H-labeled estrone or 17beta-estradiol, was taken up by membrane vesicles from K562/BCRP cells, and this was ATP-dependent. Additionally, BCRP inhibitors suppressed the transport of estrone sulfate in membrane vesicles from K562/BCRP cells. These results suggest that BCRP does not transport either free estrone or 17beta-estradiol but exports sulfate conjugates of these estrogens.

Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization.

Breast cancer resistance protein (BCRP) is a half-molecule ABC transporter highly expressed in mitoxantrone-resistant cells. In our study we established PA317 transfectants expressing Myc-tagged BCRP (MycBCRP) or HA-tagged BCRP (HABCRP). The exogenous BCRP protein migrated as a 70-kDa protein in SDS-PAGE under reducing condition, but migrated as a 140-kDa complex in the absence of reducing agents. The 140-kDa BCRP complex was heat-stable but dissociated into 70-kDa BCRP with the addition of 2-mercaptoethanol. The 140-kDa BCRP complex was immunoprecipitated with anti-Myc antibody from the lysates of PA317 cells double-transfected with MycBCRP and HABCRP. The 140-kDa complex reacted with anti-HA and anti-BCRP antibodies and after the addition of reducing agents, a 70-kDa protein reacting with anti-Myc, anti-HA and anti-BCRP antibodies was detected. These results clearly indicate that BCRP forms a homodimer bridged by disulfide bonds. To assess the possible dominant-negative inhibition of BCRP drug efflux pump, various mutant BCRP cDNAs were isolated by PCR mutagenesis. First, mutant BCRP cDNAs were introduced to parental PA317 cells and tested for their function as drug-resistance genes. Next, inactive BCRP cDNA clones were introduced to MycBCRP-transfected cells and tested for the ability to lower drug resistance. Among the 8 inactive mutant cDNA clones tested, HABCRP cDNA clone 15 with an amino acid change from Leu to Pro at residue 554 in the fifth transmembrane domain of BCRP partially reversed the drug resistance of MycBCRP-transfected cells. These results suggest that homodimer formation is essential for BCRP drug resistance, implicating this dominant-negative inhibition as a new strategy to circumvent drug resistance.