A single proteolytic cleavage within the lower hinge of trastuzumab reduces immune effector function and in vivo efficacy.

INTRODUCTION: Recent studies reported that human IgG antibodies are susceptible to specific proteolytic cleavage in their lower hinge region, and the hinge cleavage results in a loss of Fc-mediated effector functions. Trastuzumab is a humanized IgG1 therapeutic monoclonal antibody for the treatment of HER2-overexpressing breast cancers, and its mechanisms of action consist of inhibition of HER2 signaling and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). The objective of this study is to investigate the potential effect of proteinase hinge cleavage on the efficacy of trastuzumab using both a breast cancer cell culture method and an in vivo mouse xenograft tumor model. METHODS: Trastuzumab antibody was incubated with a panel of human matrix metalloproteinases, and proteolytic cleavage in the lower hinge region was detected using both western blotting and mass spectrometry. Single hinge cleaved trastuzumab (scIgG-T) was purified and evaluated for its ability to mediate ADCC and inhibition of breast cancer cell proliferation in vitro as well as anti-tumor efficacy in the mouse xenograft tumor model. Infiltrated immune cells were detected in tumor tissues by immunohistochemistry. RESULTS: scIgG-T retains HER2 antigen binding activity and inhibits HER2-mediated downstream signaling and cell proliferation in vitro when compared with the intact trastuzumab. However, scIgG-T lost Fc-mediated ADCC activity in vitro, and had significantly reduced anti-tumor efficacy in a mouse xenograft tumor model. Immunohistochemistry showed reduced immune cell infiltration in tumor tissues treated with scIgG-T when compared with those treated with the intact trastuzumab, which is consistent with the decreased ADCC mediated by scIgG-T in vitro. CONCLUSION: Trastuzumab can be cleaved by matrix metalloproteinases within the lower hinge. scIgG-T exhibited a significantly reduced anti-tumor efficacy in vivo due to the weakened immune effector function such as ADCC. The results suggest that the lower hinge cleavage of trastuzumab can occur in the tumor microenvironment where matrix metalloproteinases often have high levels of expression and scIgG-T might compromise its anti-tumor efficacy in the clinic. However, further studies are needed to validate these hypotheses in the clinical setting.

Elevated albumin in retinas of monkeys with experimental glaucoma.

PURPOSE: To establish the identity of a prominent protein, approximately 70 kDa, that is markedly increased in the retina of monkeys with experimental glaucoma compared with the fellow control retina, the relationship to glaucoma severity, and its localization in the retina. METHODS: Retinal extracts were subjected to 2-D gel electrophoresis to identify differentially expressed proteins. Purified peptides from the abundant 70 kDa protein were analyzed and identified by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) separation, and collision-induced dissociation sequencing. Protein identity was performed on MASCOT (Matrix Science, Boston, MA) and confirmed by Western blot. The relationship between the increase in this protein and glaucoma severity was investigated by regression analyses. Protein localization in retina was evaluated by immunohistochemistry with confocal imaging. RESULTS: The abundant protein was identified as Macaca mulatta serum albumin precursor (67 kDa) from eight non-overlapping proteolytic fragments, and the identity was confirmed by Western blot. The average increase in retinal albumin content was 2.3 fold (P = 0.015). In glaucoma eyes, albumin was localized to some neurons of the inner nuclear layer, in the inner plexiform layer, and along the vitreal surface, but it was only found in blood vessels in control retinas. CONCLUSIONS: Albumin is the abundant protein found in the glaucomatous monkey retinas. The increased albumin is primarily localized to the inner retina where oxidative damage associated with experimental glaucoma is known to be prominent. Since albumin is a major antioxidant, the increase of albumin in the retinas of eyes with experimental glaucoma may serve to protect the retina against oxidative damage.

Urine proteome analysis in murine nephrotoxic serum nephritis.

BACKGROUND: Urine contains serum proteins filtered by the glomerulus or secreted by the renal tubules and proteins produced locally by the urinary tract. Proteomic analysis of urine holds the potential as a noninvasive means of studying or monitoring disease activity. In mice, large concentrations of albumin and lipocalins have complicated the ability to identify urinary biomarkers in disease models. METHODS: Passive nephrotoxic serum nephritis was induced in mice. Urine proteins were identified and quantified by iTRAQ and MALDI-TOF mass spectrometry. Results were compared to Western blotting and multiplex immunoassays. RESULTS: Large concentrations of major urinary proteins dominate the urine proteome of mice even in the context of acute nephritis. Increased proteinuria caused by nephrotoxic serum nephritis is transient and includes increased albumin excretion. There were no alterations in chemokine excretion. Altered hepcidin excretion was identified, most likely reflecting local production and renal retention. CONCLUSION: Proteomic analysis of mouse urine remains challenging due to the abundance of a limited subset of proteins. iTRAQ analysis does not circumvent these challenges, but can provide information on post-translational processing of some proteins. Hepcidin is identified as a potential urinary marker of nephritis and its role in disease pathogenesis warrants further study.

Salivary biomarkers for the detection of malignant tumors that are remote from the oral cavity.

Proteomic analyses by mass spectrometry are propelling the field of medical diagnostics forward at unprecedented rates because of its ability reliably to identify proteins that are at the femtomole level in concentration. These advancements have also benefited biomarker research to the point where saliva is now recognized as an excellent diagnostic medium for the detection of malignant tumors that are remote from the oral cavity. Saliva is easy to collect and may provide diagnostic information about a variety of cancers. In particular, proof-of-principle has been demonstrated for salivary biomarker research. This article reviews the literature, discusses the theories associated with saliva-based tumor diagnostics, and presents the current research focused on the use of saliva as a diagnostic medium for the detection of cancer.

A Comparison of the Proteomic Expression in Pooled Saliva Specimens from Individuals Diagnosed with Ductal Carcinoma of the Breast with and without Lymph Node Involvement.

Purpose. The objective was to compare the salivary protein profiles of saliva specimens from individuals diagnosed with invasive ductal carcinoma of the breast (IDC) with and without lymph node involvement. Methods. Three pooled saliva specimens from women were analyzed. One pooled specimen was from healthy women; another was from women diagnosed with Stage IIa IDC and a specimen from women diagnosed with Stage IIb. The pooled samples were trypsinized and the peptide digests labeled with the appropriate iTRAQ reagent. Labeled peptides from each of the digests were combined and analyzed by reverse phase capillary chromatography on an LC-MS/MS mass spectrometer. Results. The results yielded approximately 174 differentially expressed proteins in the saliva specimens. There were 55 proteins that were common to both cancer stages in comparison to each other and healthy controls while there were 20 proteins unique to Stage IIa and 28 proteins that were unique to Stage IIb.

Determinants of human and mouse melanoma cell sensitivities to oleandrin.

Oleandrin, a cardiac glycoside component of Nerium oleander, has been shown to induce apoptosis in malignant cells. While human tumor cells are very sensitive to growth inhibition by oleandrin, murine tumor cells are extremely resistant. Using human BRO and mouse B16 melanoma cell lines, we explored several possible determinants of cell sensitivity to oleandrin and compared with ouabain. The studies include Na+, K(+)-ATPase activity and its isoforms as well as the cellular uptake of these cardiac glycosides. Oleandrin and ouabain induced apoptosis was detected in BRO cells while no evidence of cell death was observed in B16 cells even at concentrations 1000-fold higher than that used for BRO cells. Cellular uptake of oleandrin and ouabain was 3-4 fold greater in human BRO tumor cells than murine tumor cells. Partially purified Na+, K(+)-ATPase from human BRO cells was inhibited at a concentration that was 1000-fold less than that was required to inhibit mouse B16 enzyme to the same extent. Using Western blot analyses, human BRO cells were found to express both the sensitive alpha3 isoform and the less sensitive alpha1 isoform of Na+, K(+)-ATPase while mouse B16 cells expressed only the alpha1 isoform. These data suggest that differential expressions of Na+, K(+)-ATPase activities and its isoforms in BRO and B16 cells as well as cellular drug uptake may be important determinants of tumor cell sensitivity to cardiac glycosides.

Identification of serum biomarkers in brain-injured adults: potential for predicting elevated intracranial pressure.

Brain injury biomarkers may have clinical utility in stratifying injury severity level, predicting adverse secondary events or outcomes, and monitoring the effectiveness of therapeutic interventions. As a biomarker source, serum offers several advantages over cerebrospinal fluid (CSF), including ease of accessibility and reduced risk to the patient. We screened pooled serum samples obtained from 11 severely injured traumatic brain injury (TBI) patients (Glasgow Coma Scale [GCS] 25 mm Hg). Our results support the use of serum as a source for discovery of TBI biomarkers, and indicate that serum biomarkers may have utility for predicting secondary pathologies (e.g., elevated ICP) associated with TBI.

Breast cancer related proteins are present in saliva and are modulated secondary to ductal carcinoma in situ of the breast.

OBJECTIVE: The objective of this study was to determine if protein-by-products secondary to cancer related oncogenes appear in the saliva of breast cancer patients. METHODS: Three pooled (n = 10 subjects/pool) stimulated whole saliva specimens from women were analyzed. One pooled specimen was from healthy women, another pooled specimen from women diagnosed with a benign breast tumor and the other one pooled specimen was from women diagnosed with ductal carcinoma in situ (DCIS). Differential expression of proteins was measured by isotopically tagging proteins in the tumor groups and comparing them to the healthy control group. Experimentally, saliva from each of the pooled samples was trypsinized and the peptide digests labeled with the appropriate iTRAQ reagent. Labeled peptides from each of the digests were combined and analyzed by reverse phase (C18) capillary chromatography on an Applied Biosystems QStar LC-MS/MS mass spectrometer equipped with an LC-Packings HPLC. RESULTS: The results of the salivary analyses in this population of patients yielded approximately 130 proteins in the saliva specimens. Forty-nine proteins were differentially expressed between the healthy control pool and the benign and cancer patient groups. CONCLUSIONS: The study suggests that saliva is a fluid suffused with solubilized by-products of oncogenic expression and that these proteins may be modulated secondary to DCIS. Additionally, there may be salivary protein profiles that are unique to both DCIS and fibroadenoma tumors.

Regulation of gap junction coupling through the neuronal connexin Cx35 by nitric oxide and cGMP.

Gap-junctional coupling among neurons is subject to regulation by a number of neurotransmitters including nitric oxide. We studied the mechanisms by which NO regulates coupling in cells expressing Cx35, a connexin expressed in neurons throughout the central nervous system. NO donors caused potent uncoupling of HeLa cells stably transfected with Cx35. This effect was mimicked by Bay 21-4272, an activator of guanylyl cyclase. A pharmacological analysis indicated that NO-induced uncoupling involved both PKG-dependent and PKG-independent pathways. PKA was involved in both pathways, suggesting that PKG-dependent uncoupling may be indirect. In vitro, PKG phosphorylated Cx35 at three sites: Ser110, Ser276, and Ser289. A mutational analysis indicated that phosphorylation on Ser110 and Ser276, sites previously shown also to be phosphorylated by PKA, had a significant influence on regulation. Ser289 phosphorylation had very limited effects. We conclude that NO can regulate coupling through Cx35 and that regulation is indirect in HeLa cells.

Proteomic profiling of urinary protein excretion in the factor H-deficient mouse.

BACKGROUND: Since the 1970s a variety of experimental techniques have been employed in an attempt to identify urinary biomarkers of renal injury. While these approaches have met with some success, modern proteomic tools now permit broad based high-throughput analysis of the urinary proteome. METHODS: Using the ICAT isotopic labeling based LC/MS/MS approach, comparative urinary protein profiling was performed in a murine model of membranoproliferative glomerulonephritis. Paired samples were analyzed mice with a targeted deletion of the complement regulatory protein factor H (FH-/-) and control mice. RESULTS: 25 distinct urinary proteins were identified of which 7 were differentially expressed in the FH-/- mice. Two proteins were markedly altered in the urine of FH-/- mice compared to controls: uromodulin (5.5-fold lower) and the MHC class II molecule H2e (8.6-fold higher). Differential expression was confirmed by Western blot and RT-PCR. Immunofluorescent staining demonstrated a marked increased expression of H2e and a reduction of uromodulin expression in the tubular epithelium of FH-/- mice. CONCLUSIONS: These findings provide insight into early complement-dependent alterations in tubular protein expression which may play critical roles in the development of tubulointerstitial disease, and provide experimental support for the use of urinary proteomic profiling in murine models of renal injury.

Nitric oxide-dependent negative feedback of PARP-1 trans-activation of the inducible nitric-oxide synthase gene.

Nitric oxide (NO) participates in a variety of physiologic and pathophysiologic processes in diverse tissues, including the kidney. Although mechanisms for cytokine induction of inducible nitric-oxide synthase (iNOS) have been increasingly clarified, the controls for termination of NO production remain unclear. Because excessive NO production can be cytotoxic to host cells, feedback inhibition of iNOS transcription would represent a means of cytoprotection. Many of the cGMP-independent functions of NO are mediated by S-nitrosylation of cysteine thiols of target proteins. We hypothesized that NO-mediated S-nitrosylation of transcription factors might serve to feedback inhibit their trans-activation potential and deactivate iNOS gene transcription. Transient transfection of murine mesangial cells with iNOS promoter deletion-luciferase constructs revealed the region -915 to -849 to be NO sensitive with respect to IL-1beta-induced promoter activity. In vitro DNase I footprinting identified a footprint at -865/-842 in the absence of NO, but not in the presence of endogenous or exogenously delivered NO. Southwestern blotting using this probe coupled with partial peptide sequencing of the protein bands revealed that poly(ADP-ribose) polymerase isoform 1 (PARP-1) bound the probe in a sequence-specific manner. Gel shift/supershift experiments and chromatin immunoprecipitation assay analysis confirmed this binding in vitro and in vivo. Functionally, mutation of the -859/-850 site to prevent PARP-1 binding or PARP-1 knockdown by RNA interference relieved the inhibitory effects of NO on iNOS promoter activity. Biotin-switch assays and co-immunoprecipitation with an anti-nitrocysteine antibody indicated that PARP-1 was S-nitrosylated. We conclude that NO feedback inhibits iNOS gene transcription by S-nitrosylating the trans-activator PARP-1 and decreasing its binding and/or action at the iNOS promoter.