Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migration of human melanoma cells and elicits PTEN elevation and inactivation of PI3K/AKT signaling.

BACKGROUND: The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity. Herein we have extended these studies to the effects of ODAM on cultured melanoma cell lines. METHODS: The A375 and C8161 melanoma cell lines were stably transfected with ODAM and assayed for properties associated with tumorigenicity including cell growth, motility, and extracellular matrix adhesion. In addition, ODAM-transfected cells were assayed for signal transduction via AKT which promotes cell proliferation and survival in many neoplasms. RESULTS: ODAM expression in A375 and C8161 cells strongly inhibited cell growth and motility in vitro, increased cell adhesion to extracellular matrix, and yielded significant cytoskeletal/morphologic rearrangement. Furthermore, AKT activity was downregulated by ODAM expression while an increase was noted in expression of the PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor gene, an antagonist of AKT activation. Increased PTEN in ODAM-expressing cells was associated with increases in PTEN mRNA levels and de novo protein synthesis. Silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing melanoma cells. Similar PTEN elevation and inhibition of AKT by ODAM was observed in MDA-MB-231 breast cancer cells while ODAM expression had no effect in PTEN-deficient BT-549 breast cancer cells. CONCLUSIONS: The apparent anti-neoplastic effects of ODAM in cultured melanoma and breast cancer cells are associated with increased PTEN expression, and suppression of AKT activity. This association should serve to clarify the clinical import of ODAM expression and any role it may serve as an indicator of tumor behavior.

ODAM Expression Inhibits Human Breast Cancer Tumorigenesis.

We have posited that Odontogenic Ameloblast Associated Protein (ODAM) serves as a novel prognostic biomarker in breast cancer and now have investigated its potential role in regulating tumor growth and metastasis. Human breast cancer MDA-MB-231 cells were transfected with a recombinant ODAM plasmid construct (or, as a control, the plasmid vector alone). ODAM expression increased adhesion and apoptosis of the transfected MDA-MB-231 cells and suppressed their growth rate, migratory activity, and capability to invade extracellular matrix-coated membranes. Implantation of such cells into mouse mammary fat pads resulted in significantly smaller tumors than occurred in animals that received control cells; furthermore, ODAM-expressing cells, when injected intravenously into mice, failed to metastasize, whereas the control-transfected counterparts produced extensive lung lesions. Our finding that induction of ODAM expression in human breast cancer cells markedly inhibited their neoplastic properties provides further evidence for the regulatory role of this molecule in tumorigenesis and, consequently, is of potential clinical import.

Inhibition of pathologic immunoglobulin-free light chain production by small interfering RNA molecules.

OBJECTIVE: Morbidity and mortality occurring in patients with multiple myeloma, AL amyloidosis, and light chain deposition disease can result from the pathologic deposition of monoclonal immunoglobulin light chains (LCs) in kidneys and other organs. To reduce synthesis of such components, therapy for these disorders typically has involved antiplasma cell agents; however, this approach is not always effective and can have adverse consequences. We have investigated another means to achieve this objective; namely, RNA interference. MATERIALS AND METHODS: SP2/O mouse myeloma cells were stably transfected with a construct encoding a λ6 LC (Wil) under control of the cytomegalovirus promoter, while λ2-producing myeloma cell line RPMI 8226 was purchased from the American Type Culture Collection (Manassas, VA, USA). Both were treated with small interfering RNA directed specifically to the V, J, or C portions of the molecules and then analyzed by enzyme-linked immunosorbent assay, flow cytometry, and real-time polymerase chain reaction. RESULTS: Transfected cells were found to constitutively express detectable quantities of messenger RNA and protein Wil and, after exposure to small interfering RNAs, an ∼ 40% reduction in messenger RNA and LC production was evidenced at 48 hours. An even greater effect was seen with the 8226 cells. CONCLUSIONS: Our results have shown that RNA interference can markedly reduce LC synthesis and provide the basis for testing the therapeutic potential of this strategy using in vivo experimental models of multiple myeloma.

Odontogenic ameloblast associated protein as a novel biomarker for human breast cancer.

Odontogenic Ameloblast Associated Protein (ODAM) is a protein isolated in ameloblasts during odontogenesis. ODAM expression was identified in breast cancer, but its significance remains unknown. The purpose of this study is to determine if ODAM expression can serve as a prognostic marker and provide information regarding treatment in human breast cancer. Breast cancer patients were identified from our tumor registry from 1993 to 2003. Archived breast cancer tissue from 243 patients (stage 0 = 53, stage I = 51, stage II = 53, stage III = 47, stage IV = 39) was stained using monoclonal antibody for ODAM. Presence or absence of immunostaining was correlated with stage, histologic grade, response to chemotherapy, and survival using chi2 and logistic regression analyses. Tumor nuclear staining for ODAM increased with increasing group stage (P < 0.001). Staining for ODAM did not correlate with histologic grade or chemotherapy (P = 0.558, P = 0.093). Improved outcomes within each stage were noted with ODAM staining, statistically significant for stages 0, I, and II (P < 0.001, P = 0.003, P = 0.003) and underpowered for stages III and IV (P = 0.724, P = 0.059). Survival benefit associated with tumor nuclear staining increased with advancing stage (P < 0.001). These results show that ODAM predicts survival in breast cancer. Research is ongoing to determine ODAM's clinical utility and role in carcinogenesis.

AA amyloidosis associated with a mutated serum amyloid A4 protein.

AA amyloidosis invariably has been associated with fibrillar deposits of the acute phase high-density lipoprotein serum amyloid A isotypes SAA1 and SAA2. We now report the first case in a patient with no antecedent history of a chronic inflammatory or neoplastic process whose pathologic renal deposits were comprised of a mutated form of the constitutively expressed serum amyloid A4 (SAA4) protein. Analyses by tandem mass spectrometry of amyloid extracted from kidney biopsies revealed a component identical in sequence to the N-terminal portion of SAA4, except for the substitution of glycine for tryptophan at position 22 (W22G). Sequencing of genomic DNA using SAA4-specific primers showed a TGG to GGG transversion in codon 22 that accounted for the observed modification. Confirmation of the SAA4 nature of the amyloid was obtained immunohistochemically. Notably, only wild-type SAA4 was detected by mass spectrometry in the patient's serum and its concentration was within normal limits. Given the substitution of an amino acid lacking a side chain for a bulky residue, we posit that the W22G alteration would profoundly affect SAA4 stability, rendering it amyloidogenic. Our studies provide the first evidence for a novel type of AA amyloidosis in which the fibrils were formed from a mutated SAA4 protein.

Expression of odontogenic ameloblast-associated protein (ODAM) in dental and other epithelial neoplasms.

We previously have communicated our discovery that the amyloid associated with calcifying epithelial odontogenic tumors is composed of N-terminal fragments of the structurally novel odontogenic ameloblast-associated protein designated ODAM. Subsequently, it was shown by other investigators that ODAM is expressed in rodent enamel organ and is likely involved in dental development. We now report that this molecule also is found in certain human tissues, principally the salivary gland and trachea, as evidenced by RNA array analysis and immunohistochemistry-utilizing antibodies prepared against synthetic ODAM-related peptides and recombinant protein. Notably, these reagents immunostained normal and malignant ameloblasts and other types of human neoplastic cells, including those of gastric, lung, and breast origin where the presence in the latter was confirmed by in situ hybridization using gene-specific molecular probes. Moreover, significant titers of anti-ODAM IgG antibodies were detected in the sera of patients with these malignancies. Our studies have provided the first evidence in humans for the cellular expression of ODAM in normal and diseased states. Based on our findings, we posit that ODAM is a developmental antigen that has an essential role in tooth maturation and in the pathogenesis of certain odontogenic and other epithelial neoplasms; further, we suggest that ODAM may serve as a novel prognostic biomarker, as well as a potential diagnostic and therapeutic target for patients with breast and other epithelial forms of cancer.

Odontogenic ameloblast-associated protein nature of the amyloid found in calcifying epithelial odontogenic tumors and unerupted tooth follicles.

We have previously reported that the amyloid found in three patients with calcifying epithelial odontogenic tumors (CEOT) was composed of N-terminal fragments of a putative 153-residue protein specified by a gene designated FLJ20513 now known to represent exons 5 through 10 of the odontogenic ameloblast-associated protein (ODAM) locus that encodes a 279-residue polypeptide. Confirmation of the amyloidogenic potential of ODAM has resulted from analyses of four other cases where we found, in addition, a 74-residue segment specified by exon 4. Through preparation of ODAM-related synthetic peptides, it was possible to localize the fibril-forming region of this molecule, as well as generate a monoclonal antibody that reacted specifically with the amyloid associated with CEOT. Notably, we also detected green birefringent congophilic material in unerupted tooth follicles - a precursor of CEOT - and demonstrated through immunologic and chemical analyses the ODAM nature of the deposits. Our studies have provided further evidence for this unique form of odontogenic amyloid that we provisionally designate "AODAM".

Regulation of apoptosis in eicosapentaenoic acid-treated HL-60 cells.

BACKGROUND: Neutrophil apoptosis is an important physiological process in the resolution of pulmonary inflammation. Previous studies have shown that eicosapentaenoic acid (EPA; 20:5n-3) increases the rate of apoptosis in a concentration- and time-dependent manner in HL-60 cells. However, it is not known if the EPA-induced apoptosis involves the lipoxygenase (LO) and cyclooxygenase (COX) enzymes or the downstream metabolic products of these enzymes. Thus, the objective of this study was to determine the effects of inhibitors LO and COX enzymes on apoptosis, viability, and necrosis in EPA-treated HL-60 cells. MATERIALS AND METHODS: Cells were incubated with 50 mum EPA in the presence of an enzyme inhibitor (1-10 microm) for 12 h. Compounds were used to inhibit COX 1 and 2 (ibuprofen), 5-, 12-, 15-LO (NDGA), 12-LO (baicalein), 5-LO (AA-861), and 5-LO activating protein (MK-886). Eicosanoid (0.001-1.0 mum) add-back experiments were also conducted; LTB(4) and 5-HETE with 5-LO inhibition and 12-HETE with 12-LO inhibition. Flow cytometry was used to assess apoptosis. RESULTS: Inhibition of COX 1 and 2 had no effect on apoptosis. Inhibition of 5-LO and 12-LO significantly increased apoptosis in EPA-treated HL-60 cells. Addition of LTB(4) reduced apoptosis to levels significantly lower than in HL-60 cells treated with EPA alone; 5-HETE and 12-HETE also lowered apoptosis to control levels. CONCLUSIONS: These data indicate that inhibition of LO, particularly 5-LO, increased apoptosis in EPA-treated HL-60 cells. Furthermore, this study demonstrated that the products of the LO enzymes, particularly LTB(4), are critical in the regulation of apoptosis in EPA-treated HL-60 cells.

Effects of siRNAs in combination with Gleevec on K-562 cell proliferation and Bcr-Abl expression.

RNA interference (RNAi) is the repression of gene expression through a cellular mechanism of transcript-specific mRNA degradation. RNAi has been observed in human cells and applied to the modulation of a variety of human transcripts. Our goals were to deliver small interfering RNA (siRNA) using a liposome-based method, and to show Bcr-Abl siRNA specificity against K-562 cells, alone or in combination with Gleevec. Both synthetic (syn) siRNA, consisting of homogeneous 21-nucleotide-long RNA duplexes specific for the Bcr-Abl fusion site, and recombinant (r)-generated Bcr-Abl siRNA were employed. siRNA was transfected into K-562 cells with greater than 90% efficiency using RNAiFect, as judged by fluorescence analysis. The Bcr-Abl transcript was inhibited using either siRNA preparation as measured by RT-PCR or real-time PCR. The IC(50) of Gleevec in the K-562 subline F(1) was lowered over 3-fold from 0.2 to 0.06 muM in cells transfected with either syn or rBcr-Abl siRNA. No effect was observed in cells after transfection with an irrelevant control siRNA. Therefore, K-562 cells transfected with RNAifect deliver Bcr-Abl siRNA efficiently and the Bcr-Abl siRNA decreased the IC(50) of Gleevec required to inhibit the high levels of Bcr-Abl protein found in K-562 cells.

Characterization of a monoclonal antibody identifying a CD45RA antigen on feline leukocytes.

The antibody produced by a murine hybridoma obtained from the fusion of SP2/0 plasmacytoma cells with splenocytes of a mouse immunized with feline bone marrow was found to react with 60% of bone marrow cells and 80% of peripheral blood leukocytes (PBL); reactivity in the latter tissue was restricted almost entirely to mononuclear cells. Two-color FACScan analyses of this antibody with mAbs specific for feline lymphocytes revealed positive and negative populations of CD4 and CD8 cells. The reactivity for CD4 and CD8 cells was animal age dependent, binding to a higher percentage of the cells in young (2-9 months) versus older animals (> 4 years). In a mitogen driven assay for IgG production by PBL the addition of this antibody to the cultures enhanced the suppressor activity of CD8 cells, a function attributed to activation of a CD4 suppressor-inducer population; removal of CD8 cells negated any induction of suppression. Mild papain digestion of bone marrow and PBL completely removed the antigen detected by this antibody while not affecting reactivity of a pan-T antibody. Western blot analysis showed binding of the antibody to polypeptides of approximately 200 kDa on feline bone marrow and PBL. The data suggest that this mAb is identifying the feline homologue of the leukocyte common antigen of cells with a functional specificity characteristic of a CD45RA isoform.