The SUPPRESSOR OF rps4-RLD1 (SRFR1) gene was identified based on enhanced AvrRps4-triggered resistance in the naturally susceptible Arabidopsis accession RLD. No other phenotypic effects were recorded, and the extent of SRFR1 involvement in regulating effector-triggered immunity was unknown. Here we show that mutations in SRFR1 in the accession Columbia-0 (Col-0) lead to severe stunting and constitutive expression of the defense gene PR1. These phenotypes were temperature-dependent. A cross between srfr1-1 (RLD background) and srfr1-4 (Col-0) showed that stunting was caused by a recessive locus in Col-0. Mapping and targeted crosses identified the Col-0-specific resistance gene SNC1 as the locus that causes stunting. SRFR1 was proposed to function as a transcriptional repressor, and SNC1 is indeed overexpressed in srfr1-4. Interestingly, co-regulated genes in the SNC1 cluster are also upregulated in the srfr1-4 snc1-11 double mutant, indicating that the overexpression of SNC1 is not a secondary effect of constitutive defense activation. In addition, a Col-0 RPS4 mutant showed full susceptibility to bacteria expressing avrRps4 at 24°C but not at 22°C, while RLD susceptibility was not temperature-dependent. The rps4-2 snc1-11 double mutant showed increased, but not full, susceptibility at 22°C, indicating that additional cross-talk between resistance pathways may exist. Intriguingly, when transiently expressed in Nicotiana benthamiana, SRFR1, RPS4 and SNC1 are in a common protein complex in a cytoplasmic microsomal compartment. Our results highlight SRFR1 as a convergence point in at least a subset of TIR-NBS-LRR protein-mediated immunity in Arabidopsis. Based on the cross-talk evident from our results, they also suggest that reports of constitutive resistance phenotypes in Col-0 need to consider the possible involvement of SNC1.

Author Summary

Plants, like humans, have an immune system to defend against disease. This immune system seeks out the presence of disease-causing microbes and other invaders by detecting non-plant molecules and proteins. Plants rely on this surveillance to activate an antimicrobial response of appropriate strength at the right time; as with humans, an overactive immune system can be harmful to plants. We study how plants achieve an appropriate balance, using genetics and the interaction between the reference plant Arabidopsis thaliana and the bacterial plant pathogen Pseudomonas syringae. So-called plant resistance proteins are important activators of immunity that directly or indirectly intercept foreign proteins deployed by pathogens. Resistance proteins are generally thought to be highly specific detectors that only respond to a single pathogen protein. However, while working with a negative regulator of plant immunity called SRFR1, we discovered a surprising level of cross-talk between different resistance proteins that becomes evident only under certain environmental conditions such as low temperature. We also show that SRFR1 and these resistance proteins bind to each other, possibly explaining the observed cross-talk. Our work thus highlights linkages between resistance pathways and provides insight into the molecular architecture of the plant innate immune response.

Reduced growth and viability is a common phenotype of plants with constitutively activated pathogen defenses. One branch of the plant innate immunity system, effector-triggered immunity, is especially potent and requires tight control to enable normal plant development. While some facets of this control that directly regulate resistance protein abundance or activity have been documented, general control of effector-triggered signaling sensitivity is poorly understood. We recently identified SUPPRESSOR OF rps4-RLD 1 (SRFR1), a novel negative regulator of avrRps4-triggered immunity. Mutations in SRFR1 were previously shown not to induce constitutive high expression of the defense gene PR1, and to be fully susceptible to the virulent Pseudomonas syringae pv. tomato strain DC3000. SRFR1 encodes a tetratricopeptide repeat-containing protein with weak similarity to transcriptional repressors in other organisms. By transient expression in Nicotiana benthamiana, SRFR1 was localized to the nucleus. Here we investigate more carefully whether expression of defense genes is misregulated in srfr1 mutant plants. Consistent with the hypothesized function of SRFR1 as a negative transcriptional regulator, we find that mRNA levels of several defense genes are upregulated in srfr1 mutants.

EC-18 (monoacetyldiacylglyceride) stimulates T cell production of IL-2, IL-4, IL-12, IFN-γ, and GM-CSF in vitro. To study the effects of these cytokines stimulated by EC-18 on cancer cells, we applied hamster biliary cancer model, a difficult cancer to treat. Cancer (KIGB-5) cells were given intravenously to produce hematogenous metastatic lung lesions which were treated with EC-18 at 10, 25, and 50 mg/kg/day respectively. The fourth group was untreated control. At 4th, 8th, and 12th week the lungs were examined. EC-18 treated groups showed only a few microscopic lung lesions and no evidence of metastatic lesion with highest dose whereas widespread gross lung lesions were observed in untreated control. To investigate whether the anti-tumor effect of EC-18 is associated with suppression of tumor cell Toll-like receptor 4 (TLR-4) expression in addition to stimulation of the immune cells, KIGB-5 cells were exposed to LPS with or without EC-18. TLR-4 mRNA and protein expression, measured by reverse transcriptase PCR (RT-PCR), real-time quantitative PCR and western blot analysis, showed suppression of TLR-4 expression in KIGB-5 cells treated with EC-18 compared with control. In conclusion, EC-18 has a significant anti-tumor effect in this experimental model of biliary cancer suggesting potential for clinical application to this difficult cancer.

Transplantation of marrow-derived mesenchymal stem cells (MSCs), expanded by culture in addition to whole bone marrow, has been shown to enhance engraftment of human hematopoietic stem cells (HSCs). Our hypothesis was that there might be an optimum ratio range that could enhance engraftment. We examined the percent donor chimerism according to the ratio of HSCs to MSCs in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We tested a series of ratios of co-transplanted CD34+-selected bone marrow cells, and marrow-derived MSCs into sublethally irradiated NOD/SCID mice. In all experiments, 1×105 bone marrow derived human CD34+ cells were administered to each mouse and human MSCs from different donors were infused concomitantly. We repeated the procedure three times and evaluated engraftment with flow cytometry four weeks after each transplantation. Serial ratios of HSCs to MSCs were 1:0, 1:1, 1:2 and 1:4, in the first experiment, 1:0, 1:1, 1:2, 1:4 and 1:8 in the second and 1:0, 1:1, 1:4, 1:8 and 1:16 in the third. Cotransplantation of HSCs and MSCs enhanced engraftment as the dose of MSCs increased. Our results suggest that the optimal ratio of HSCs and MSCs for cotransplantation might be in the range of 1:8-1:16; whereas, an excessive dose of MSCs might decrease engraftment efficiency.

Purpose

Autologous stem cell transplantation (ASCT) is increasingly used in patients with non-Hodgkin's lymphoma (NHL). Various clinical parameters-were evaluated to obtain significant predictors of the outcome following ASCT in patients with NHL.

Materials and Methods

Between April 1994 and December 2003, ASCT was performed on 80 patients with NHL at the Asan Medical Center.

Results

Patients had various histological subtypes and disease status. The two year progression free survival (PFS) and overall survival for all patients were 34 and 31%, respectively. A univariate analysis showed the performance status, stage, modified extranodal involvement category, International Prognostic Index (IPI) at mobilization, disease status at mobilization, and history of radiation prior to mobilization as significant predictors of the outcome following ASCT. Four risk groups, with different 2 year PFS, were identified by the age adjusted IPI at mobilization (mAAIPI): low risk 44%; low intermediate risk 40%; high intermediate risk 19%; and high risk 0% (p=.0003). A multivariate analysis revealed 3 significant factors for the PFS: disease status, prior RT and mAAIPI.

Conclusion

The mAAIPI was found to be an independent predictor of the outcome of NHL patients undergoing ASCT. This powerful prognostic tool should be used to evaluate potential candidates for ASCT.

Purpose

The benefit of consolidation high-dose chemotherapy (HDC) for high-risk primary breast cancer is controversial. We evaluated the efficacy and safety of consolidation HDC with cyclophosphamide, thiotepa and carboplatin (CTCb) followed by autologous stem-cell transplantation (ASCT) in resected breast cancer patients with 10 or more positive lymph nodes.

Materials and Methods

Between December 1994 and April 2000, 22 patients were enrolled. All patients received 2 to 6 cycles of adjuvant chemotherapy after surgery for breast cancer. The HDC regimen consisted of cyclophosphamide 1,500 mg/m2/day, thiotepa 125 mg/m2/day and carboplatin 200 mg/m2/day intravenous for 4 consecutive days.

Results

With a median follow-up of 58 months, 11 patients recurred and died. The median disease-free survival (DFS) and median overall survival (OS) were 49 and 69 months, respectively. The 5-year DFS and OS rates were 50% and 58%, respectively. The 12 patients with 10 to 18 involved nodes had better 5-year DFS (67%) and OS (75%) than 10 patients with more than 18 involved nodes (30% and 38%, respectively). The most common grade 3 or 4 nonhematologic toxicity was diarrhea, which occurred in 5 patients (23%). No treatment-related death was observed.

Conclusion

Consolidation HDC with CTCb followed by ASCT for resected breast cancer with more than 10 positive nodes had an acceptable toxicity but does not show promising survival.

Purpose

The benefit of high-dose chemotherapy (HDC) for metastatic breast cancer (MBC) is controversial. We evaluated the efficacy and safety of HDC with cyclophosphamide, thiotepa, and carboplatin (CTCb) followed by autologous stem-cell transplantation (ASCT) for MBC patients.

Materials and Methods

From September 1994 to December 1999, 23 MBC patients were enrolled. All the patients received 2 to 10 cycles of induction chemotherapy. Before transplantation, 12 patients were in complete response (CR), nine were in partial response (PR), and two had progressive disease (PD). The HDC regimen consisted of cyclophosphamide 1,500 mg/m2/day, thiotepa 125 mg/m2/day and carboplatin 200 mg/m2/day intravenously for 4 consecutive days.

Results

After ASCT, 13 patients (56%) had a CR, five (22%) had a PR, three (13%) had no change, while two (9%) showed a PD. Seventeen patients relapsed or progressed during the median follow-up of 78 months. The median progression-free survival (PFS) time was 11 months and the median overall survival (OS) time was 23 months. The 5-year PFS and OS rates were 22% and 25%, respectively. On the multivariate analyses, less than 4 involved lymph nodes was predictive of a better PFS and OS.

Conclusion

HDC with CTCb for MBC has acceptable toxicity; however, this treatment does not show a survival benefit.

Purpose

A constituent of green tea, (-)-epigallocatechin-3-gallate (EGCG), is known to possess anti-cancer properties. In this study, the time-course of the anticancer effects of EGCG on human ovarian cancer cells were investigated to provide insights into the molecular-level understanding of the growth suppression mechanism involved in EGCG-mediated apoptosis and cell cycle arrest.

Materials and Methods

Three human ovarian cancer cell lines (p53 negative, SKOV-3 cells; mutant type p53, OVCAR-3 cells; and wild type p53, PA-1 cells) were used. The effect of EGCG treatment was studied via a cell count assay, cell cycle analysis, FACS, Western blot and macroarray assay.

Results

EGCG exerts a significant role in suppressing ovarian cancer cell growth, showed dose dependent growth inhibitory effects in each cell line and induced apoptosis and cell cycle arrest. The cell cycle was arrested at the G1 phase by EGCG in SKOV-3 and OVCAR-3 cells.

In contrast, the cell cycle was arrested in the G1/S phase in PA-1 cells. EGCG differentially regulated the expression of genes and proteins (Bax, p21, Retinoblastoma, cyclin D1, CDK4 and Bcl-XL) more than 2 fold, showing a possible gene regulatory role for EGCG. The continual expression in p21WAF1 suggests that EGCG acts in the same way with p53 proteins to facilitate apoptosis after EGCG treatment. Bax, PCNA and Bcl-X are also important in EGCG-mediated apoptosis. In contrast, CDK4 and Rb are not important in ovarian cancer cell growth inhibition.

Conclusion

EGCG can inhibit ovarian cancer cell growth through the induction of apoptosis and cell cycle arrest, as well as in the regulation of cell cycle related proteins. Therefore, EGCG-mediated apoptosis could be applied to an advanced strategy in the development of a potential drug against ovarian cancer.

Dendritic cells (DCs) are potent antigen-presenting cells for the induction and activation of cytotoxic T lymphocytes. We tested whether bone marrow-derived DCs are capable of inducing protective immunity against a murine lymphoma (A20). DCs were grown from tumor-bearing BALB/c mice by culturing bone marrow cells. BALB/c mice were injected (sc) with A20 cells on day 0. Intraperitoneal immunization with DCs mixed with lethally irradiated A20 cells were started when the tumor reached ca. 4-5 mm in diameter (Group A) or on day -7 (Group B). Booster immunizations were given every 3-4 days for four weeks. By 31 days in group A, there was a significant reduction in tumor growth in the mice immunized with DCs mixed with irradiated A20 cells as compared with the control groups (p=0.016). In group B, tumor growth was completely inhibited and there was no tumor growth following extended observations after completion of immunization. Thus, DCs mixed with irradiated tumor cells can induce an antitumor effect. This provides a rationale for the use of DCs mixed with irradiated tumor cells in immunotherapy for minimal residual disease of lymphomas.

Treatment-related myelodysplastic syndrome (t-MDS) and acute myelogenous leukemia (t-AML) are now well established as complications of cytotoxic chemotherapy. We experienced a 28-yr-old female patient who developed t-MDS/t-AML with characteristic chromosomal abnormalities including 11q23 chromosomal rearrangement following high-dose chemotherapy with autologous stem cell transplantation (ASCT) for non-Hodgkin's lymphoma. The patient was admitted with bulky abdominal masses of B cell lineage non-Hodgkin's lymphoma. After 2 cycles of systemic chemotherapy of the Vanderbilt regimen, the patient underwent ASCT with high dose chemotherapy of the BEAC regimen. She also received radiation of 48 Gy for the residual periportal lymphadenopathy. The initial cytogenetic analysis of the infused mononuclear cells revealed a normal karyotype. Twenty two months after the ASCT, pancytopenia was noted and her bone marrow aspirate showed dysplastic hemopoiesis with myeloblasts up to 12% of nonerythroid nucleated cells. The patient was diagnosed as t-MDS (refractory anemia with an excess of blasts). Cytogenetic analysis showed complex chromosomal abnormalities including 11q23 rearrangement, which is frequently found in topoisomerase II inhibitor-related hematologic malignancies. Four months later, it was noted that the t-MDS had evolved into an overt t-AML. Cytogenetic analysis showed an evolving pattern with more complex abnormalities. The patient was treated with combination chemotherapy, but her leukemic cells were resistant to the therapy.