Biomedical subjects
Publications and source records attributed to Robert E Brown.
Blastic NK cell lymphoma is a rare hematolymphoid neoplasm. This report illustrates an unusual presentation of this entity, namely as a primary leukemia, but without skin lesions.
Head and neck squamous cell carcinoma (HNSCC) has a relatively high mortality rate and poor prognosis. Recently, we showed that overexpression of phosphorylated (p) nuclear factor-kappaB (NF-kappaB) in squamous cell carcinoma of the tonsil (SCCT) and high grade dysplasia is associated with a poor prognosis. Because the mammalian target of the rapamycin (mTOR) pathway contributes to the activation of NF-kappaB through immunophilin/mTOR signaling, we investigated: (a) the immunohistochemical expression and state of activation and potential clinical significance of components of the mTOR signal transduction pathway in SCCT patients (morphoproteomics); and (b) the inhibitory effects of rapamycin on the growth and state of activation of mTOR in 2 HNSCC cell lines (pharmacoproteomics). Archival biopsy materials from 39 patients with SCCT were studied by immunohistochemistry for the expression of p-mTOR (Ser 2448), and p-p70S6K (Thr 389), and/or cyclin D1. Results for SCCT were compared with adjacent non-neoplastic epithelium, when present, and with normal tonsillar epithelium from approximately age-matched controls; clinical outcomes were also assessed. SCCT showed mTOR (Ser 2448) expression in 93% (30/32 cases) with 2+ or 3+ plasmalemmal and/or cytoplasmic intensity in 84% vs 42% in surface epithelium from normal tonsils (p <0.001). The mean combined expression score (signal intensity x percentage of positive cells) for p-p70S6K was significantly greater in the SCCT group vs adjacent non-neoplastic squamous epithelium and normal tonsillar epithelium of the control group (p <0.05). A relationship existed between higher p-p70S6K expression levels in the non-neoplastic squamous epithelium adjacent to the SCCT and increased risk of death from disease (hazard ratio = 7.9; 95% confidence interval (CI) = 2.1 to 29.9; p = 0.002). There was also a relationship between nuclear expression of cyclin D1 in SCCT and shortened recurrence-free survival (p = 0.015). Two human HNSCC cell lines, SCC-15 and FaDu, were incubated with and without rapamycin to assess its impact on growth and on the expression of p-mTOR. Rapamycin in a dose-dependent fashion inhibited growth more in SCC-15, which correlated with a greater reduction in constitutively activated p-mTOR (Ser 2448) as shown by Western blotting. In conclusion, these morphoproteomic and pharmacoproteomic data collectively provide a rationale for selecting mTOR effectors as therapeutic targets in HNSCC.
CCI-779 (temsirolimus), an ester of rapamycin and an inhibitor of the mammalian target of rapamycin (mTOR), is currently in phase II trials for treatment of patients with solid cancers. The mTOR functions as a checkpoint for cell proliferation, with upstream Akt and downstream p70S6K serving as its most important mediators. The aim of this study was to evaluate the expression and activation of the Akt-mTOR-p70S6K pathway in renal cell carcinoma (RCC), seeking to strengthen the rationale for targeted therapy of RCC using rapamycin or a rapamycin analogue. Tissue microarray sections containing 128 primary RCCs, 22 metastatic RCCs, and 24 non-neoplastic (normal) kidneys (NK) were immunostained with monoclonal antibodies to phosphorylated (p)-Akt (Ser473), p-mTOR (Ser2448), and p-p70S6K (Thr389). Western blotting was performed on 3 cases of clear cell RCC (CRCC) and the corresponding non-neoplastic (normal) renal tissues using the same antibodies. The immunostain scoring system included: (a) location; (b) distribution; and (c) intensity. The normal kidneys provided baseline scores for comparison. Expression of p-Akt, p-mTOR, and p-p70S6K was seen in 100% (n = 24) of NKs and nearly 100% (n = 150) of both primary and metastatic RCCs. The p-p70S6K was located in the nucleus in both NKs and RCCs. The p-Akt was observed in the nucleus and cytoplasm of NKs and in the nucleus and cytoplasm/ membrane (plasmalemma) of RCCs. The p-mTOR was identified in the membrane of NKs and the membrane/nucleus of RCCs. The levels of expression of p-p70S6K, p-mTOR, and p-Akt were significantly higher in RCC than in NK in the overall pattern (intensity and distribution, p <0.05). Western blotting also showed higher expression of p-p70S6K, p-mTOR, and p-Akt in RCCs compared to the corresponding normal kidney tissues (p <0.05). These findings indicate that correlative over-expression and activation of p-Akt, p-mTOR, and p-p70S6K are commonly observed in RCCs. After considering these findings in the context of other established protein circuitries and pathways in RCC, we propose therapeutic approaches that incorporate rapamycin-like agents and other small molecule inhibitors in a combinatorial fashion in future clinical trials for RCC.
Glioblastoma multiforme (GBM) presents a major challenge to neurosurgeons, neuro-oncologists, and radiation therapists by virtue of its location with a blood-brain barrier, chemoradioresistance, highly malignant phenotype, and angiogenic potential. Because nuclear factor-kappaB (NF-kappaB) can transcriptionally activate genes leading to the synthesis of anti-apoptotic, chemoresistant, growth promoting, and angiogenic proteins; we assessed the state of activation of NF-kappaB in 6 GBM cases at diagnosis. Morphoproteomic analysis confirmed the constitutive activation of NF-kappaB by demonstrating the phosphorylation (p) and nuclear translocation of p-NF-kappaBp65 (Ser 536) in these cases. This observation coincides with (a) previous immunohistochemical findings showing nuclear translocation of total p65, (b) demonstration of NF-kappaB DNA binding activity, (c) the results of electrophoretic mobility shift assays, and (d) existing genomic data in GBM. Furthermore, such constitutive activation of the NF-kappaB pathway helps to explain some of the tumor biology and supports the incorporation of NF-kappaB pathway inhibitors into the treatment of GBM.
Intracellular signals along the epidermal growth factor receptor (EGFR)-Akt-nuclear factor-kappa B (NF-kappaB) pathway have been associated with carcinogenesis in various malignant neoplasms. This investigation was to evaluate the expression of EGFR, phosphorylated(p)-Akt and p-NF-kappaB and correlate them with clinical outcomes in patients with squamous cell carcinoma of the tonsil. A total of 45 patients with squamous cell carcinoma of the tonsil were studied by immunohistochemistry to evaluate the expression levels of EGFR, p-Akt and p-NF-kappaB. Results for squamous cell carcinoma of the tonsil were compared with those for associated high-grade dysplasia and adjacent normal appearing epithelium, when present. In addition, tonsillar epithelium from non-neoplastic specimens of age-matched patients also was stained for the same markers. High-grade dysplasia and squamous cell carcinoma of the tonsil demonstrated a similar pattern of expression, which differed from the pattern seen in the adjacent normal epithelium and tonsillar epithelium from normal controls (an overexpression for each of these three protein analytes in high-grade dysplasia and squamous cell carcinoma of the tonsil as demonstrated by immunohistochemistry). When markers from squamous cell carcinoma of the tonsil were correlated with survival status, only increasing levels of p-NF-kappaB immunoreactivity (a relative overexpression) were statistically significant predictors of poor survival. No markers in squamous cell carcinoma of the tonsil were significantly related to rate of recurrence. When analyzing marker scores from tissue with high-grade dysplasia, relative overexpressions of both p-Akt and p-NF-kappaB were significantly related to poor survival. Additionally, increasing levels of p-NF-kappaB immunopositivity from tissue with high-grade dysplasia were also significantly related to rate of recurrence. In summary, p-NF-kappaB, overexpressed in high-grade dysplasia and squamous cell carcinoma of the tonsil, is associated with worse prognosis in terms of high recurrence and poor survival, respectively. This significant finding in patients with squamous cell carcinoma of the tonsil, in combination with previous animal and in vitro studies, suggests that p-NF-kappaB represents a potential therapeutic target in head and neck squamous cell carcinoma.
Campath-1H has been used successfully for induction and has resulted in a low rate of acute cellular rejection (ACR) in renal transplantation in combination with various postoperative immunosuppression regimens. This study was undertaken to investigate the extent of monocyte involvement in ACR, with or without Campath-1H induction. We found that monocytes represented the majority of inflammatory cells in grades Ib or higher ACR, but not with Ia type of ACR, regardless of the status of Campath-1H induction. Cases of ACR, following Campath-1H induction, appear to demonstrate a 'pure form' of monocytic ACR, whereas monocytes were mixed with many other types of inflammatory cells in the cases of ACR in the absence of Campath-1H induction. In addition with Campath-1H induction, the cases of monocyte-predominant ACR were found to uniformly exhibit a good response to corticosteroid treatment. We conclude that monocyte-predominate ACR may represent a severe form of rejection, with or without Campath-1H treatment.
Morphoproteomics combines the disciplines of histopathology, molecular biology and protein chemistry to paint a portrait of the protein circuitry in diseased cells for the purpose of uncovering molecular targets amenable to specific intervention, thereby customizing therapy for individual patients. This review considers the clinical application of morphoproteomics in malignant cells in the context of currently available pharmaceutical agents and discusses opportunities for combinatorial approaches that involve one or more small molecule inhibitors and single-agent chemotherapy with relatively low toxicity profiles. Future directions that involve focusing on points of convergence in signal transduction pathways and which integrate morphoproteomic with genomic and pharmacoproteomic and protein-function microarray data are offered.
Velcade, a proteasome inhibitor, has been shown to inhibit DNA binding activity of nuclear factor-kappaB (NF-kappaB) and to stabilize p53 in vitro. But its impact, in the context of activated (phosphorylated and translocated) NF-kappaB and the expression of p53, has not been studied in breast cancer. It would be desirable to determine whether or not the immunohistochemical (IHC) expressions of activated NF-kappaB and of p53 can predict the effects of Velcade in viable tumor cells. To answer these questions, we selected 3 breast cancer cell lines (SKBR-3, MDA-175, and MDA-231), which are negative for hormonal receptors, but differ in HER-2/neu expression (strong, mild, and minimal, respectively). The 3 cell lines showed different expressions of phosphorylated (p)- NF-kappaB and p53, as evaluated using immunohistochemistry with visual quantification by brightfield microscopy. After being treated with Velcade for 2 days, MDA-231 cells showed markedly reduced proliferation, followed by SKBR-3 cells, and then by MDA-175 cells. There was strong correlation between the nuclear expression of either p-NF-kappaB or p53 and the inhibitory rate of Velcade in the 3 cell lines (r = 0.987 and 0.807, respectively). Western blotting showed an increase in inhibitor-kappaB (I-kappaB) expression in nuclei of MDA-231 and SKBR-3 cells, but not in MDA-175 cells, following exposure to Velcade. Velcade treatment resulted in cleaved caspase-3 expression in MDA-231 cells and in the overexpression of p53 and p21WAF1 in all 3 cell lines, as evaluated using Western blotting. In summary, morphoproteomic analysis of p-NF-kappaB and p53 can be correlated with the inhibitory effect of Velcade in vitro. We propose that this proliferative inhibition is variably associated with blocking p-NF-kappaB function by upregulation of nuclear I-kappaB, stabilization of p53, and induction of p21WAF1.
Morphoproteomics utilizes immunohistochemistry to identify protein analytes in tumor cells in order to uncover or confirm potential molecular pathways that may be essential to their proliferation, integrity, and histogenesis and that may serve as therapeutic targets. This communication illustrates the application of such an approach to osteolytic Langerhans cell histiocytosis and considers such molecular targets in the context of currently available therapies.
BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is synthesized in the cytoplasm, complexed with its inhibitor, I-kappaB, and NF-kappaB is released in an activated (phosphorylated) form following phosphorylation of I-kappaB and proteasomal degradation of the NF-kappaB.p-kappaB complex. The free p-NF-kappaB can then be translocated to the nucleus where it effects transcriptional activation of genes leading to the synthesis of proteins that are generally pro-growth and anti-apoptosis. OBJECTIVE: To gain insight into the role of the NF-kappaB pathway in head and neck squamous cell carcinoma (HNSCC), we selected two HNSCC cell lines, SCC-15 of lingual origin and FaDu of pharyngeal origin, with constitutively activated (phosphorylated) NF-kappaB. We assessed the impact of interrupting the NF-kappaB pathway at the level of proteasomal degradation using Velcade (bortezomib), a proteasome inhibitor, and at the pretranslational level in the synthesis of NF-kappaB using a small interfering RNA (siRNA). RESULTS: Velcade produced a dose-dependent inhibition of cell growth in both cell lines. At 30 nM, Velcade inhibited cell growth in the SCC-15 cell line by 40%. In both cell lines, Velcade induced nuclear overexpression of p21(WAF1), an inhibitor of G1 cell cycle progression, which appeared to be independent of p53 expression. Addition of siRNA augmented the inhibitory effects of Velcade in FaDu cells; siRNA/NF-kappaB alone led to a 48% decline in basal expression of NF-kappaB protein levels and effected a 25% inhibition of cell growth. In the presence of Velcade (30 nM), siRNA/NF-kappaB increased growth inhibition from 43 to 65%. CONCLUSIONS: The mechanisms involved in growth inhibitory effects of Velcade on HNSCC cell lines include the NF-kappaB pathway, suggesting the possible therapeutic use of Velcade or other NF-kappaB pathway inhibitors (eg, curcumin). The data also suggest that combining siRNA/NF-kappaB with Velcade might achieve greater reduction in the growth of HNSCC in patients with constitutively activated NF-kappaB.
Platelet-derived growth factor receptor-alpha (PDGFR-alpha) expression in the Hodgkin/Reed-Sternberg cells of nodular sclerosing Hodgkin's disease has been demonstrated in 2 studies. Additional receptor tyrosine kinases have also now been documented. This communication reports the correlative expression in nodular sclerosing Hodgkin's disease of activated (phosphorylated) p70S6K, one of the putative downstream effectors common to receptor tyrosine kinase signaling.
P504S/alpha-methylacryl CoA racemase has been shown to be a relatively sensitive and specific positive marker for prostatic adenocarcinoma. The potential utility of P504S in renal cell neoplasms has not been explored in a large series. We assessed the diagnostic value of P504S in 332 cases of nonprostatic neoplasms using the avidin-biotin-peroxidase complex technique, including 115 renal neoplasms, 28 metastatic renal cell carcinomas (RCCs), and 189 nonrenal neoplasms. The results demonstrated that a granular, cytoplasmic staining pattern for P504S was observed in 48 of 70 (68.6%) conventional (clear cell) RCCs, 15 of 15 (100%) papillary RCCs, 2 of 7 (29%) chromophobe RCCs, and 2 of 8 (25%) oncocytomas. Among the 70 cases of clear cell RCC, positivity of P504S was seen in 40%, 71%, 94%, and 75% of RCCs with Furhman nuclear grade I, II, III, and IV, respectively. Strong immunostaining was present in each case (86/86) in the proximal tubules adjacent to the renal neoplasm. Eighty-two percent of metastatic RCCs (23/28) were positive for P504S. However, only 24 of 189 (13%) nonrenal malignancies were positive. The 24 positive cases included 12 of 13 (92%) colorectal adenocarcinomas, 6 of 30 (20%) ductal carcinomas of the breast, and 4 of 23 (17%) adenocarcinomas of the lung. These findings suggest that P504S is a useful marker in diagnosing primary and metastatic RCCs, although it has little value in differentiating chromophobe RCC from oncocytoma.
Specialized equipment, changes to procedures, non-entry by design, and customized/creative approaches can eliminate or reduce the frequency of the need to enter confined spaces. Consider the new safety hazards that may be created, but always remember: The safest entry is no entry at all!
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"Morphoproteomics" is defined as the identification by immunohistochemistry of the molecular circuitry of various proteins in a tumor by noting their state of activation (translocation and phosphorylation) and correlative expressions. This approach can uncover potential targets, amenable to therapeutic interventions, that are specific for an individual patient's tumor (ie, customized chemotherapy). This communication illustrates the successful application of such an approach in a patient with relapsed acute lymphoblastic leukemia.
Campath-1H (alemtuzumab), a humanized monoclonal antibody against CD52, can cause more profound depletion of lymphocytes than monocytes. The resultant imbalance of lymphocytes and monocytes after Campath-1H treatment of a renal-transplant recipient may lead to an acute rejection dominated by monocytes. We report such a case of acute transplant rejection in a 49-yr-old man who received a living non-related kidney transplant and was treated with preoperative Campath-1H and postoperative immunosuppression. An initial post-transplant renal biopsy showed diffuse mild acute rejection with 95% CD68-positive monocytes, but only 5% CD3-positive T lymphocytes. Inflammatory cells in the renal biopsy were negative for CD34 and CD1a stains, suggesting non-involvement of CD34-derived dendritic cells in the acute rejection. After steroid treatment for 2 wk, the patient's serum creatinine concentration diminished to 1.5 mg/dl. The histopathological features of acute rejection were absent in a second biopsy of the transplanted kidney. In summary, this case is an instance of monocyte-mediated acute rejection of a transplanted kidney.
The aim of this study was to elucidate protein circuitry in breast cancer based on the profiling of hormone-receptor-negative breast carcinomas using morphoproteomic and pharmacoproteomic techniques. Three human breast carcinoma cell lines (SKBR-3, MDA-175, MDA-231) were reacted by immunohistochemical (IHC) procedures to detect several categories of protein analytes. Immunoreactivities and cell compartmentalizations were scored from 0 to 3+ positivity using bright-field microscopy. An automated cellular imaging system (ACIS) was used to obtain a final combined score of staining intensity and positive cells in the IHC reactions, to enable comparisons with the visual scores and the rates of inhibition by pharmaceutical agents. FDA-approved inhibitors that target the protein circuitry were added to the cultures for 2-4 days. Proliferation assays were conducted, and in vitro inhibition rates were calculated as (control - treated)/control. Western blot analyses of whole cell lysates assessed the effects of the pharmaceutical agents on selected aspects of protein circuitry. Good to excellent correlation was observed between visual scores and ACIS scores (r values from 0.732 to 0.996 in 10 of 11 trials). Gleevec produced growth inhibition that correlated with the composite expressions of the platelet-derived growth factor (PDGF) family of ligands and receptors; captopril inhibited only MDA-175, consistent with its unique expression of plasmalemmal angiotensin-converting enzyme (ACE); and interferon (IFN)-alpha effected growth inhibition in accordance with the degree of conventional (c) protein kinase C (PKC)-alpha and phosphorylated (p)-PKCalpha/betaII expressions. Western blot analyses revealed correlative changes of several intracellular signals following incubation with these inhibitors. This study shows (a) a close association between the immunohistochemical expression of signal transduction markers and in vitro inhibition by pharmaceutical agents, and (b) correlations between the sites of action of the pharmaceutical agents and the downstream expression of proteins in hormone-receptor-negative breast cancer cell lines. Such morphoproteomic and pharmacoproteomic profiling of individual tumors may enable the pathologist and oncologist to design antitumor therapy that is customized for an individual patient.
To identify markers sensitive to inhibitors of the farnesylation pathway, we used 3 breast cancer cell lines (SKBR-3, MDA-175, and MDA-231) to evaluate the in vitro effects of pamidronate, an inhibitor of farnesyl diphosphate synthase. In response to pamidronate, there was significant inhibition of cell proliferation in MDA-231 and SKBR-3 cells, compared to MDA-175 cells. This correlated with their respective basal levels of N-ras and H-ras. N-ras and H-ras protein levels were both reduced in MDA-231 cells, and to lesser extent in SKBR-3 cells, following exposure to pamidronate, whereas these markers were not altered in MDA-175 cells. Combinatorial therapy with pamidronate and Gleevec, an inhibitor of several tyrosine kinases; Velcade, a proteasome inhibitor; or rapamycin, an inhibitor of the mammalian target of rapamycin (m-TOR) all showed additive effects in causing proliferative inhibition in MDA-175 cells. In summary, resistance to pamidronate may result from low levels of GTPase-activating proteins, such as N-ras and H-ras, in tumor cells. Combinatorial therapies directed against other signaling pathways, not dependent upon ras, may be required to overcome such resistance.