Daniel P. Kestler

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.

Leukocyte Chemotactic Factor 2 (LECT2)-Associated Renal Amyloidosis: A Case Series

BACKGROUND Renal amyloidosis is characterized by the pathologic deposition within glomeruli and/or interstitium of congophilic fibrils, most often composed of either immunoglobulin light chains or serum amyloid A-related protein and, less commonly, mutated forms of apolipoproteins AI or AII, lysozyme, fibrinogen, gelsolin, or transthyretin. STUDY DESIGN Case series. SETTING & PARTICIPANTS 10 patients with renal amyloidosis who had an amyloidogenic protein that was not identified using routine immunohistochemistry. OUTCOMES Clinical, pathologic, biochemical, and genetic characteristics. MEASUREMENTS Tandem mass spectrometry was used to analyze fibrils extracted from sections of formalin-fixed paraffin-embedded amyloid-containing kidney biopsy specimen blocks. RESULTS Chemical analyses showed peptides corresponding to the carboxy-terminal portion of the leukocyte chemotactic factor 2 (LECT2) molecule. In addition, deposits were immunostained using an anti-human LECT2 monoclonal antibody. Plasma specimens were available from 2 individuals for whom LECT2 concentration in these samples was within the reference range. Additionally, in 4 of the cases analyzed at the molecular level, isolation of genomic DNA and polymerase chain reaction amplification of LECT2-encoding exons showed no mutations. However, all were homozygous for the G allele encoding valine at position 40 in the mature protein, a finding confirmed using restriction enzyme analysis of the polymorphic site. LIMITATIONS Causality is not addressed. CONCLUSIONS Based on our studies, we posit that LECT2-associated renal amyloidosis represents a unique and perhaps not uncommon disease, especially in Mexican Americans. The pathogenesis, extent, and prognosis remain to be determined.

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”.

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.

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.

Hematopoietic differentiation activity of a recombinant human interleukin-6 (IL-6) isoform resulting from alternatively spliced deletion of the second exon

We have previously identified and cloned an alternatively spliced form of human interleukin‐6 mRNA lacking exon II, which encodes amino acid residues known to be important in gp130‐mediated signal transduction pathways. We expressed and purified the recombinant protein (rIL6‐alt) resulting from this alternatively spliced mRNA and now report the initial characterization of its biologic activities with comparison to full length IL6 (rIL6‐full). rIL6‐alt was found to have 104 to 105 fold less activity in proliferation assays with 7TD1 murine plasmacytoma cells and did not competitively inhibit the stimulatory activity of rIL6‐full. In addition, like rIL6‐full, rIL6‐alt had antiproliferative activity toward M1 murine myeloblast cells and was 10–200‐fold less active than rIL6‐full. In contrast, in assays with human HL60 promyelocytic leukemia cells, rIL6‐alt had greater antiproliferative activity than rIL6‐full and more strongly upregulated phagocytosis as well as surface expression of the differentiation antigen CD11b. rIL6‐full and rIL6‐alt upregulated the level of lysozyme mRNA in HL60 cells approximately equally. These findings suggest that IL6‐alt, which lacks amino acid residues encoded by the second exon of the gene, is not a natural inhibitor of IL6‐full but may be relatively tissue specific and may play a role in modulation of hematopoietic cell growth and differentiation. Am. J. Hematol. 61:169–177, 1999.

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

BackgroundThe 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.MethodsThe 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.ResultsODAM 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.ConclusionsThe 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.

Leukocyte chemotactic factor 2 (LECT2)-associated renal amyloidosis.

In substantiation of a recent account of a patient whose renal amyloid deposits were formed from leukocyte chemotactic factor 2 (LECT2), we report 10 additional cases in adults ranging in age from 58 to 84 years who exhibited varying degrees of renal functional impairment and proteinuria. Analyses by tandem mass spectrometry of amyloid extracted from sections cut from formalin-fixed, paraffin-embedded blocks revealed, after trypsin digestion, peptides corresponding to the carboxyterminal portion of the LECT2 molecule; further, the deposits were immunostained by an anti-human LECT2 monoclonal antibody. In four samples analyzed at the molecular level, no mutations in the LECT2-encoding exons were revealed. Based on our studies, we posit that LECT2-associated amyloidosis (ALECT2) represents a unique and perhaps not uncommon disease, the pathogenesis, extent, and prognosis of which remain unknown. Introduction: Renal amyloidosis results from the pathologic deposition as fibrils in glomeruli and/or parenchyma of monoclonal immunoglobulin light chains or serum amyloid A protein, as well as apolipoproteins AI and AII, fibrinogen, lysozyme, gelsolin, and transthyretin (AL, AA, AApo AI, AApo AII, AFib, ALys, AGel, and ATTR, respectively) [1]. Recently, Benson et al. [2] discovered yet another renal amyloid protein – leukocyte chemotactic factor 2 (LECT2) – after chemical analysis of fibrils isolated from a surgically resected clear cell cancer-containing kidney from a 61-year-old woman. We now report the discovery of 10 additional examples of LECT2 renal amyloidosis in adults. Methods: Amyloid extracted from formalin-fixed, paraffin-embedded renal biopsies was reduced, carboxymethylated, and digested with trypsin under conditions previously described [3,4]. The resultant peptides were subjected to mass spectrometry (MS/ MS). Immunohistochemistry was performed using a goat anti-human LECT2 monoclonal antibody, followed by exposure to a biotinylated anti-goat IgG reagent, and then to streptavidin HRP. The reaction was developed with ImmPACT diaminobenzidine. Blood LECT2 concentrations were determined colorimetrically, as per the manufacturer’s protocol, using the Ab-Match ASSEMBLY and UNIVERSAL Human LECT2 kits, kindly furnished by Dr. Satoshi Yamagoe. The primers and conditions used for genomic DNA amplification by polymerase chain reaction of protein encoding exons 2, 3, and 4 of Figure 1. LECT2-associated renal amyloid. (Left) Extensive birefringent congophilic interstitial (and minimal glomerular) deposits immunostained with (Right) an anti-human LECT2 antiserum (original magnifications, 61000). 223

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 patients 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.

Non-hereditary apolipoprotein AI-associated pulmonary amyloid

Apolipoprotein AI amyloidosis (Apo AI) characteristically is associated with the deposition in tissue of fibrils composed of mutated Apo AI; however, amyloid deposits comprised of the native molecules have been noted in the aorta of elderly humans. We now report that an amyloid-containing nodule in the lung of a 76-year-old asymptomatic female was composed of Apo AI that, on the basis of genomic DNA analysis, was formed from wild-type protein. This finding provides further evidence that the aging process is associated with the propensity of normally soluble unmutated serum proteins to deposit pathologically as amyloid. Introduction: Typically, the apolipoprotein (Apo) AI-associated amyloidoses are autosomal, dominant, familial disorders characterized by the pathologic deposition as fibrils of N-terminal fragments of mutated Apo AI molecules in the nerves, kidney, liver, heart, or other organs [1]. Notably, however, inheritable variations in Apo AI sequence may not necessarily render these components amyloidogenic, as demonstrated by the discovery that the Apo AIrelated deposits seen in the aortic intima of older humans (as well as the pulmonary vasculature of aged dogs) is comprised of wild-type protein [2,3]. We now report our analysis of amyloid contained in a 1.6 cm lung nodule (Figure 1) from an otherwise healthy, non-smoking, 76-year-old Caucasian female, which represented an amyloidoma composed of unmutated Apo AI. Methods: Amyloid extracted from sections cut from a formalin-fixed, paraffin-embedded CT-guided needle biopsy specimen was reduced, alkylated, digested with trypsin, and subjected to tandem mass spectrometry (MS/MS) [4,5]. For molecular analysis, genomic DNA was prepared from peripheral blood leukocytes and the 700 base pairs comprising exon 4 of the Apo AI gene were amplified by PCR using primers and conditions, as already published [6]; the products were sequenced at the University of Tennessee’s Molecular Biology Facility. The procedures for immunohistochemistry, using as primary reagent a murine anti-human Apo AI mAb, were as previously described [7]. Results and discussion: Analyses by MS/MS of the peptides resulting from trypsin digestion of the reduced and alkylated amyloid extracted from the biopsy specimen revealed components that spanned residues 24–40 and 62–77 of normal Apo AI; further, no mutations were found in Apo AI-encoding exons of the patient’s genomic DNA. The Apo AI nature of the amyloid was confirmed immunohistochemically (Figure 1). Our data provide evidence for yet another manifestation of Apo AI-related disease in an elderly individual in whom the pulmonary amyloid deposit consisted of wild-type protein. Acknowledgements: Authors thank Sallie Macy and Craig Wooliver for the studies illustrated in Figure 1. Declaration of interest: Research was supported, in part, by United States Public Health Service Figure 1. (a,b) CT scan of the lung showing the presence of the amyloidoma. (a) Bone window with increased contrast; (b) Soft tissue window. (c,d) Congo red and immunohistochemical analysis of the same section of the patient’s lung. (c) Congo red stain; (d) Immunostaining of the amyloid by an anti-Apo AI antibody (ABC method). Original magnifications, 6200. 219