C. Darrell Jennings

Cutting Edge: Constitutive B Cell Receptor Signaling Is Critical for Basal Growth of B Lymphoma

B lymphomas account for the majority of the lymphoma cases. BCR expression appears to be important for B lymphoma because most oncogenes are translocated to nonrearranged Ig loci and because all of the variants that arise in anti-idiotypic Ab-treated lymphoma patients remain BCR positive. Based on this and the fact that BCR is required for mature B cell survival, we tested the requirement for continued expression of BCR for the growth and survival of B lymphoma cells. Using Igα or Igβ-specific small interfering RNA (siRNA) to inhibit BCR expression, we demonstrate for the first time that constitutive signaling by BCR is critical for survival and proliferation of both murine and human B lymphoma cells. The BCR signals in lymphoma appear to be mediated by Syk, as it is constitutively active in a variety of B lymphoma cells. Blocking Syk activity by selective inhibitors suppresses growth of several murine and human B lymphomas.

Spleen Tyrosine Kinase (Syk), a Novel Target of Curcumin, Is Required for B Lymphoma Growth

Curcumin (diferuloylmethane), a component of dietary spice turmeric (Curcuma longa), has been shown in recent studies to have therapeutic potential in the treatment of cancer, diabetes, arthritis, and osteoporosis. We investigated the ability of curcumin to modulate the growth of B lymphomas. Curcumin inhibited the growth of both murine and human B lymphoma in vitro and murine B lymphoma in vivo. We also demonstrate that curcumin-mediated growth inhibition of B lymphoma is through inhibition of the survival kinase Akt and its key target Bad. However, in vitro kinase assays show that Akt is not a direct target of curcumin. We identified a novel target for curcumin in B lymphoma viz spleen tyrosine kinase (Syk). Syk is constitutively activated in primary tumors and B lymphoma cell lines and curcumin down-modulates Syk activity accompanied by down-regulation of Akt activation. Moreover, we show that overexpression of Akt, a target of Syk, or Bcl-xL, a target of Akt can overcome curcumin-induced apoptosis of B lymphoma cells. These observations suggest a novel growth promoting role for Syk in lymphoma cells.

Association of HFE mutations with neurodegeneration and oxidative stress in Alzheimer's disease and correlation with APOE

Oxidative stress enhanced by transition metals such as iron forms an attractive hypothesis for neurodegeneration in Alzheimers Disease (AD). Iron is increased in the brain in AD, but whether this is a primary abnormality or the result of secondary accumulation is unclear. Among several genetic loci associated with AD, the locus at chromosome 6p21 contains the hereditary hemochromatosis gene HFE. To determine whether a genetic predisposition to iron accumulation is associated with AD, we evaluated three hemochromatosis‐associated HFE mutations and APOE in cognitively and histopathologically evaluated subjects with AD, mild cognitive impairment (MCI), non‐demented controls with AD‐like pathologic changes defined by Braak stage ≥ 3 (high pathology controls (HPC)), and non‐demented controls without significant histologic changes (low‐pathology controls (LPC)). In a subset, we examined ventricular (CSF) fluid F2‐isoprostane (F2‐IsoP) levels, a marker of lipid peroxidation. Seventeen subjects demonstrated homozygous or compound heterozygous HFE mutations, 13 (9.4%) in the AD/MCI group (P = 0.019 vs. LPC) and four (20%) in the HPC group (P = 0.006, P < 0.05 with Bonferroni correction vs. LPC). In contrast, the APOE4 allele frequency was increased only in the AD/MCI patients (P < 10−3 vs. HPC, P < 10−6 vs. LPC). F2‐IsoP levels were increased in AD subjects with any HFE mutation versus wild type HFE (P = 0.027). Although confirmation is required, these findings suggest that HFE mutations are associated with increased oxidative stress and Braak stage, and that HFE and APOE genotypes are different between AD patients, high pathology and low pathology controls.

Analysis of blood T‐cell cytokine expression in B‐chronic lymphocytic leukaemia: evidence for increased levels of cytoplasmic IL‐4 in resting and activated CD8 T cells

The cytoplasmic cytokines of purified blood T cells (CD4/C]D8) in B‐CLL patients (n = 5) and controls (n =5) were evaluated by flow cytometry. The mean levels of cytoplasmic IL‐4 were significantly elevated in resting and activated B‐CLL CD8 cells compared to control CD8 cells. IL‐4 cytoplasmic levels were comparable for resting B‐CLL and control CD4 cells but greater for B‐CLL activated CD4 cells. The mean fluorescence intensity of B‐CLL CD8 cytoplasmic IL‐4 was 4–5‐fold greater, indicating higher IL‐4 density per CLL CD8 than control CD8 cells. Both CLL CD4 and CD8 cells post‐activation had higher levels of cells double positive for cytoplasmic IL‐4 and interferon. These data indicate that freshly isolated CD8 and CD4 blood T cells from B‐CLL patients have significantly elevated (above control) levels of commitment to expression of IL‐4. Since IL‐4 has an important modulatory impact on CLL and normal B cells, this observation has implications regarding the biology of B‐CLL.

Cancer Resistance in Transgenic Mice Expressing the SAC Module of Par-4

Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that induces apoptosis in cancer cells, but not in normal/immortalized cells. The cancer-specific proapoptotic action of Par-4 is encoded in its centrally located SAC domain. We report here the characterization of a novel mouse model with ubiquitous expression of the SAC domain. Although SAC transgenic mice displayed normal development and life span, they were resistant to the growth of spontaneous, as well as oncogene-induced, autochthonous tumors. Resistance to tumorigenesis was linked to inhibition of nuclear factor-kappaB activity and induction of apoptosis by the SAC domain. Collectively, our findings provide genetic evidence that the SAC domain of Par-4 confers cancer resistance in transgenic mice without compromising normal viability or aging, and may have therapeutic significance.

Involvement of DNA mismatch repair in folate deficiency-induced apoptosis☆

Folate is a critical factor for DNA metabolism and its deficiency is associated with a number of human diseases and cancers. Although it has been shown that folate deficiency induces genomic instability and apoptotic cell death, the underlying mechanism is largely unknown. Given the role of mismatch repair in maintaining genomic integrity, mismatch repair was tested for its involvement in folate deficiency-induced genomic instability and cell death. Cells proficient in mismatch repair were highly sensitive to folate deficiency compared with cells defective in either hMutSalpha or hMutLalpha. Since wild-type cells but not mutant cells underwent apoptosis upon extensive folate depletion, the apoptotic response is dependent on a functional mismatch repair system. Our data also indicate that p53 is required for the folate depletion-induced apoptosis. In vitro biochemical studies demonstrated that hMutSalpha specifically recognized DNA damage induced by folate deficiency, suggesting a direct participation of mismatch repair proteins in mediating the apoptotic response. We conclude that while the mismatch repair-dependent apoptosis is necessary to protect damaged cells from tumorigenesis, it may damage a whole tissue or organ, as seen in patients with megaloblastic anemia, during extensive folate deficiency.

Preferential loss of mismatch repair function in refractory and relapsed acute myeloid leukemia: potential contribution to AML progression

Acute myeloid leukemia (AML) is an aggressive hematological cancer. Despite therapeutic regimens that lead to complete remission, the vast majority of patients undergo relapse. The molecular mechanisms underlying AML development and relapse remain incompletely defined. To explore whether loss of DNA mismatch repair (MMR) function is involved in AML, we screened two key MMR genes, MSH2 and MLH1, for mutations and promoter hypermethylation in leukemia specimens from 53 AML patients and blood from 17 non-cancer controls. We show here that whereas no amino acid alteration or promoter hypermethylation was detected in all control samples, 18 AML patients exhibited either mutations in MMR genes or hypermethylation in the MLH1 promoter. In vitro functional MMR analysis revealed that almost all the mutations analyzed resulted in loss of MMR function. MMR defects were significantly more frequent in patients with refractory or relapsed AML compared with newly diagnosed patients. These observations suggest for the first time that the loss of MMR function is associated with refractory and relapsed AML and may contribute to disease pathogenesis.

Anomalous constitutive Src kinase activity promotes B lymphoma survival and growth

BackgroundPreviously we have shown that B cell receptor (BCR) expression and B cell receptor signaling pathways are important for the basal growth of B lymphoma cells. In particular we have shown that the activation of Syk, a non-src family protein tyrosine kinase and the mitogen activated protein kinases (MAPK), ERK and JNK that mediate BCR signals are required for the constitutive growth of B lymphoma cells. Since src family protein tyrosine kinases (SFKs) like Lyn are known to be needed for the phosphorylation of BCR co-receptors, Ig-α and Ig-β, we hypothesized that one or more SFKs will be constitutively activated in B lymphoma cells and may be necessary for B lymphoma growth.ResultsSrc kinase activity was found to be constitutively high in many murine and human B lymphoma cell lines and primary lymphoma samples. The specific pharmacological inhibitors of SFKs, PP1 and PP2 inhibited the proliferation of a number of both murine and human B lymphomas in a dose-dependent manner. Importantly, dasatinib (BMS-354825), an oral dual BCR-ABL and SFK specific inhibitor inhibited the growth of B lymphomas in the nanomolar range in vitro and strongly inhibited a mouse lymphoma growth in vivo. Among the SFKs, Lyn is predominantly phosphorylated and Lyn-specific small interfering RNA inhibited the growth of B lymphomas, supporting an important role for Lyn in B lymphoma growth. Suppression of SFK activity blocks BCR mediated signaling pathways. PMA or CpG can partially reverse the growth inhibition induced by SFK inhibition. Although blocking SFK activity inhibited the growth of a number of B lymphomas, some lymphomas such as SudHL-4, SudHL-6, OCI-Ly3 and OCI-Ly10 are more resistant due to an increased expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL.ConclusionsThese studies further support our concept that BCR signaling pathways are important for the continued growth of established B lymphoma cells. Some of the intermediates in this BCR pathway are potential immunotherapeutic targets. In particular, inhibition of SFK activity alone or in synergy with inhibition of the prosurvival Bcl-2 proteins holds promise in developing more effective treatments for B lymphoma patients.

Flow Cytometry: Recent Advances in Diagnosis and Monitoring of Leukemia

Immunophenotyping of leukemias by flow cytometry is the result of two significant basic science innovations of the previous two decades: monoclonal antibodies and advances in laser and computer technology leading to the availability of high-quality flow cytometers.

Proliferating cell nuclear antigen in prostatic adenocarcinoma: Correlation with established prognostic indicators

OBJECTIVE The utility of an antibody to proliferating cell nuclear antigen (PCNA), a growth-specific nuclear protein, was assessed as a prognostic variable for prostatic adenocarcinoma. Its expression was correlated with established prognostic indicators, including tumor grade, stage, prostatic-specific antigen (PSA), and percent of tumor in the gland at excision. METHODS Forty archival needle biopsies containing a minimum of four hundred tumor cells were analyzed. Immunoperoxidase staining of paraffin sections was performed for PCNA (PC10) after pretreatment in antigen retrieval solution. A proliferative index (PI) for each case was derived using image analysis with measurement of at least four hundred twenty-five nuclei. RESULTS PI values ranged from 2.4 to 31.3 percent. Mean PI values varied significantly (ANOVA, p = 0.005) among cases with dominant Gleason grade (DGG) of 3 (mean PI = 9.3%), 4 (mean PI = 13.7%), and 5 (mean PI = 18.8%). By t test, significant differences were noted for PI in cases with DGG 2 and 3 versus those with DGG 4 and 5 (p = 0.0065). PI for cases with DGG 3 versus 5 showed significant difference (p = 0.0017). Tumors of Gleason scores 5 to 7 differed significantly from those with scores 8 to 10 (p = 0.014). A statistical relationship for PI and PSA, clinical stage, and percent tumor at resection could not be established by linear regression. CONCLUSIONS These findings suggest that additional study of the PI, as determined by PCNA immunohistochemistry and image analysis, may be warranted to determine its usefulness as an adjunctive parameter in prostate adenocarcinoma. This technique may be particularly useful in needle biopsies where limited tumor may render assessment of grade difficult.