Exploring Jolkinol D Derivatives To Overcome Multidrug Resistance in Cancer.

Macrocyclic monoacyl lathyrane derivatives bearing a benzoyl moiety were previously found to be strong ABCB1 modulators. To explore the effects of different substituents of the aromatic moiety, 14 new compounds (1.1-1.7, 1.10, and 2.1-2.4) were prepared from jolkinol D (1), obtained from Euphorbia piscatoria, and from jolkinodiol (2), its hydrolysis derivative. Compounds 1.8 and 1.9, having aliphatic moieties, were also obtained. The reversal of ABCB1-mediated MDR was evaluated through functional and chemosensitivity assays on the human ABCB1-gene-transfected L5178Y mouse T-lymphoma cell line. Structure-activity relationships showed that addition of electron-donating groups to the aromatic moiety improved the activity. The effects on the ATPase activity of the strongest modulator (1.3) and the inactive jolkinol D (1) were also investigated and compared. Moreover, in the chemosensitivity assay, most of the compounds interacted synergistically with doxorubicin. Compounds 1.1-1.10 and 2.1-2.4 were further assessed for their collateral sensitivity effect against the human cancer cells: EPG85-257 (gastric) and EPP85-181 (pancreatic), and the matching drug-selected cells EPG85-257RDB, EPG85-257RNOV, EPP85-181RDB, and EPP85-181RNOV. The most promising ones (1.8 and 1.10) along with compound 3, previously selected, were investigated as apoptosis inducers. The compounds were able to induce apoptosis through caspase-3 activation, with significant differences being observed between the parental and resistant cells.

BCL2 expression in CD105 positive neoangiogenic cells and tumor progression in angioimmunoblastic T-cell lymphoma.

The angiogenic microenvironment has been known to be a component of angioimmunoblastic T-cell lymphoma since its initial characterization. We have shown that angioimmunoblastic T-cell lymphoma endothelial cells produce vascular endothelial growth factor-A (VEGFA), and participate in lymphoma progression. In squamous cell carcinoma, endothelial BCL2 expression induces a crosstalk with tumor cells through VEGFA, a major mediator of tumoral angiogenesis. In the present study, we analyzed BCL2 and VEGFA in 30 angioimmunoblastic T-cell lymphomas, using triple immunofluorescence to identify protein coexpression in well-characterized lymphoma cells and microenvironment neoangiogenic endothelial cells. Using quantitative real-time PCR, we assessed mRNA expression levels in laser-microdissected endothelial and lymphoma cells. In lymphoma cells, as in endothelial cells, BCL2 and VEGFA proteins were coexpressed. BCL2 was expressed only in neoangiogenic CD34(+)CD105(+) endothelial cells. In laser-microdissected cells, mRNA studies showed a significant relationship between BCL2 and VEGFA levels in CD34(+) endothelial cells, but not in CD3(+)CD10(+)lymphoma cells, or in CD34(+) endothelial cells from lymph node hyperplasia. Further study showed that, in AITL, BCL2 mRNA levels in CD34(+)CD105(+) neoangiogenic endothelial cells also correlated with microvessel density, International Prognostic Index, Ann Arbor stage, bone marrow involvement and elevated LDH. BCL2 expression by CD105(+) neoangiogenic endothelial cells is related to tumor progression in angioimmunoblastic T-cell lymphoma.

Acute presentation of nasal-type natural killer/T-cell lymphoma of the orbit.

PURPOSE: An unusual case of nasal-type natural killer/T-cell lymphoma (NKTL) of the orbit is reported. METHODS: The clinical history, computed tomography, magnetic resonance imaging, and biopsy specimen of a 29-year-old man with a right orbital lymphoma were evaluated. RESULTS: The patient initially presented with conjunctival injection and had flu-like symptoms before developing right proptosis and reduced vision; imaging showed a diffuse infiltrative process throughout the orbit. Orbital biopsy revealed angiodestruction with prominent necrosis, and angiocentric lymphoma growth and lymphoma cells were positively stained for CD3, CD20, CD45RO, CD56, cytotoxic molecules (granzyme B and T-cell intracellular antigen-1), and Epstein-Barr virus. CONCLUSIONS: NKTL is rare and may present acutely; the imaging findings presented serve to highlight the radiologic features of the disease.

c-Maf expression in angioimmunoblastic T-cell lymphoma.

The oncogene c-Maf was recently found to be overexpressed in approximately 50% of multiple myeloma cases, and a role for c-Maf in promoting cyclin D2 expression has been postulated. We previously examined c-Maf expression in various T-cell lymphomas by reverse-transcription polymerase chain reaction and found extremely elevated c-Maf levels in angioimmunoblastic T-cell lymphoma (AILT). In this study, we examined T-cell lymphomas for c-Maf and cyclin expression immunohistochemically. Of 93 cases of T-cell lymphomas we investigated in the current study, c-Maf expression was seen in 23 out of 31 cases of AILT, 3 out of 11 of adult T-cell leukemia/lymphoma, 4 out of 19 of peripheral T-cell lymphoma, unspecified [PTCL(U)], and 0 out of 11 cases of mycosis fungoides, 0 out of 11 of anaplastic large cell lymphoma, and 1 out of 10 of extranodal NK/T-cell lymphoma, nasal type. Double immunostaining in AILT revealed that the majority of c-Maf-positive cells were also positive for CD43 (MT1), CD45RO (UCHL-1), and CD4 but were negative for CD20 (L26). Additionally, cyclins D1 and D2, which stimulate cell cycle progression, were overexpressed in a large number of the c-Maf-positive AILT samples. Quantitative reverse-transcription polymerase chain reaction analysis also showed that c-Maf was overexpressed in 8/31 cases of AILT, 0/19 cases of PTCL(U), 0/11 cases of anaplastic large cell lymphoma, 0/10 cases of extranodal NK/T-cell lymphoma, nasal type, and 2/8 cases of multiple myeloma, presenting significant difference between AILT and PTCL(U) (P=0.016, chi test). These findings strongly suggest that CD4-positive neoplastic T cells in AILT show c-Maf expression and provide new insight into the pathogenesis of AILT suggesting c-Maf to be a useful diagnostic marker for AILT.

Enteropathy-type T-cell lymphoma after intestinal diffuse large B-cell lymphoma.

A rare case of enteropathy-type T-cell lymphoma (ETL) developed in a 47-year-old Chinese male 6 years after the diagnosis of diffuse large B-cell lymphoma (DLBCL) in the small intestine. The patient initially presented with vague gastrointestinal complaints. Work-up demonstrated an ulcerated mass in the small intestine. Partial resection and histologic examination of the intestine showed a DLBCL, positive for CD20 and Bcl-2, involving the jejunum transmurally. Further staging work-up demonstrated mesenteric and retroperitoneal lymphadenopathy, splenomegaly, and ascites. The patient was treated aggressively with radiotherapy, chemotherapy, and autologous bone marrow transplant, and complete remission was obtained. Six years later, the patient presented with diarrhea and dehydration. Clinical work-up revealed thickening of the small intestinal wall, and biopsies demonstrated ETL based on morphology, immunohistochemistry, and polymerase chain reaction analysis. Celiac disease was diagnosed concurrently. The patient responded to chemotherapy, received allogeneic peripheral blood stem cell transplantation from an HLA-matched sibling donor, and remains in remission. To our best knowledge, this is the first reported case of metachronous ETL and DLBCL. Possible associations between the 2 types of lymphoma are discussed.

Hepatosplenic gammadelta T-cell lymphoma with ring chromosome 7, an isochromosome 7q equivalent clonal chromosomal aberration.

Hepatosplenic T-cell lymphoma is a rare, clinically aggressive lymphoma. Most cases represent a neoplasm of mature non-activated gammadelta T cells. Isochromosome 7q i(7)(q10) is thought to be the primary cytogenetic abnormality of this disease. In this paper, we describe a hepatosplenic gammadelta T-cell lymphoma case, with clonal ring chromosome 7 exemplifying an isochromosome 7q equivalent clonal aberration. A 62-year-old female patient presented with thrombocytopenia, isolated hepatosplenomegaly, and extremely high levels of LDH. Bone marrow work-up demonstrated a sinusoidal cytotoxic T-cell infiltrate with blastic features, while molecular studies verified monoclonal rearrangement for both TCR gamma and TCR delta genes. Cytogenetics revealed clonal abnormalities including ring chromosome 7, trisomy 8, and der(19), while FISH analysis detected 7q amplification with partial deletion of 7p in ring chromosome 7. To the best of our knowledge, this is the first reported T-cell lymphoma case with ring chromosome 7.

Involvement of CD45 in adhesion and suppression of apoptosis of mouse malignant T-lymphoma cells.

Mouse malignant T-lymphoma CS-21 cells undergo apoptotic cell death in vitro in the absence of lymph node stromal cells but escape apoptosis and proliferate when they are attached to CA-12 stromal cells. A monoclonal antibody raised against CS-21 cell surface molecules (MCS-5) recognized a M(r) 168,000 protein, inhibited binding of CS-21 cells to CA-12 stromal cells, and suppressed apoptosis in CS-21 cells. To identify the M(r) 168,000 protein, we purified it with MCS-5 affinity chromatography and ion exchange chromatography. Partial amino acid sequences of the purified M(r) 168,000 protein were identical to those of CD45, a transmembrane tyrosine phosphatase. The purified protein possessed tyrosine phosphatase activity and was recognized by an anti-CD45 monoclonal antibody. The M(r) 168,000 protein was identified as CD45. To determine the CD45 isoform, we cloned the CD45 gene from the cDNA library of CS-21. Sixteen of 18 clones encoded CD45RO (CD45 lacking exons 4, 5, and 6), and the remainder lacked exons 4, 5, 6, and 7. Like MCS-5, an anti-CD45 monoclonal antibody, also inhibited binding of CS-21 cells to CA-12 cells and suppressed apoptosis in CS-21 cells. Our present results indicate that CD45RO expressed on CS-21 cells mediates adhesion to CA-12 cells and suppression of apoptosis.

Carcinogenicity of 2',3'-dideoxycytidine in mice.

2',3'-dideoxycytidine (ddC) is a synthetic pyrimidine nucleoside analogue approved for treatment of HIV-positive patients. Previous studies indicated that ddC has the potential to cause thymic lymphoma in C57BL/6 x C3H F1 (hereafter called B6C3F1) mice. In this study, we evaluated the carcinogenic potential of ddC in two different mouse models. B6C3F1 hybrid mice carry ecotropic endogenous proviral sequences that may be activated to cause lymphoma, whereas NIH Swiss mice lack proviral sequences that can be expressed. The mice were treated with ddC by gavage at 500 and 1000 mg/kg/day for up to 6 months (human dose, 2.25 mg/day) and evaluated for toxicity, plasma levels of ddC, and pathological changes. Lymphocyte cell markers from the thymic lymphomas were assessed by immunophenotyping. Expression of p53 protein was evaluated using immunohistochemical staining. Treatment-related thymic lymphomas were present in both mouse models with a higher incidence in NIH Swiss than in B6C3F1 mice. The lymphomas were more prevalent in females than in males of both mouse models. Most mice with thymic lymphoma died during the course of the study. In addition to the thymus, lymphoma was often present in lymph nodes, spleen, and other organs. Lymphomas arose more frequently in mice that lack endogenous ecotropic retroviral sequences and thus were not due to activation of endogenous provirus. During the third month of the study, a few NIH Swiss mice that died had granulosa cell tumors of the ovary. Treatment-related but reversible thymic atrophy was observed in both mouse models. There was a very high correlation between the internal dose of ddC and the incidence of thymic lymphoma in both mouse models. Most of the lymphocytes from control thymuses and ddC-induced lymphomas were positive for Thy-1.2 (pan-T), heat stable antigen, and CD4 and CD8 markers, with no marked differences in the lymphocyte markers of the tumors between sexes or dose groups. p53 protein was detected in only 20% (23/115) of the ddC-induced lymphomas with mostly minimal expression in scattered cells. Because ddC induced lymphomas in two different mouse models, the potential carcinogenic risk should be considered in long-term treatment of HIV-positive patients, especially children and adolescent patients treated with ddC.

Effects of stem cell factor on the growth and radiation survival of tumor cells.

Recombinant human stem cell factor (SCF) binds to the c-kit receptor on human bone marrow progenitor cells and enhances their survival following irradiation. Since the c-kit receptor has also been detected on malignant cells, experiments were performed to study the effect of SCF on the proliferation and radiation survival of a variety of both c-kit-positive and -negative human tumor cell lines using [3H]thymidine incorporation and colony formation assays. The addition of SCF to both c-kit-positive and -negative cell line cultures had no significant effect on the stimulation index (in [3H]thymidine assay). In contrast, colony formation by H69 (small cell lung cancer cell line), H128 (small cell lung cancer cell line), and HEL (erythroid leukemia cell line) cells was enhanced by SCF in a dose-dependent manner, but SCF did not promote the in vivo growth of H128 xenograft tumors in terms of graft rate, time from implantation to tumor detection, or tumor size. Furthermore, SCF did not significantly increase the surviving fraction of either c-kit-positive or -negative cell lines following radiation, and there were no statistically significant differences between D0 [defined by the slope of the terminal exponential region of the two-component (single-hit multitarget model) survival curve where slope = 1/D0], Dq (quasithreshold dose), n (extrapolation number), alpha, and beta values for any of the cell lines studied that were irradiated with and without SCF. Finally, nude mice with transplanted human LG425 cutaneous T-cell lymphoma (c-kit positive) were treated with 10 Gy with or without SCF (100 micrograms/kg i.p. 20 h before, 2 h before, and 4 h after irradiation). There were no significant differences in the median tumor quadrupling time between groups that received either no treatment or SCF alone, or between groups treated with 10 Gy and SCF or 10 Gy alone (P > 0.05). These results are encouraging and suggest that SCF does not stimulate tumor cell proliferation in vivo or enhance the survival of tumor cells following irradiation.

Pathology of Extranodal Lymphoma.

An overview of the pathology of extranodal lymphoma is presented. The emphasis of this presentation is on the classification system of extranodal lymphomas, including both B-cell and T-cell lymphomas, based on their morphology, phenotype, and molecular alterations.

Re-activation of mitochondrial apoptosis inhibits T-cell lymphoma survival and treatment resistance.

T lymphocyte non-Hodgkin's lymphoma (T-NHL) represents an aggressive and largely therapy-resistant subtype of lymphoid malignancies. As deregulated apoptosis is a frequent hallmark of lymphomagenesis, we analyzed gene expression profiles and protein levels of primary human T-NHL samples for various apoptotic regulators. We identified the apoptotic regulator MCL-1 as the only pro-survival BCL-2 family member to be highly expressed throughout all human T-NHL subtypes. Functional validation of pro-survival protein members of the BCL-2 family in two independent T-NHL mouse models identified that the partial loss of Mcl-1 significantly delayed T-NHL development in vivo. Moreover, the inducible reduction of MCL-1 protein levels in lymphoma-burdened mice severely impaired the continued survival of T-NHL cells, increased their susceptibility to chemotherapeutics and delayed lymphoma progression. Lymphoma viability remained unaffected by the genetic deletion or pharmacological inhibition of all alternative BCL-2 family members. Consistent with a therapeutic window for MCL-1 treatment within the context of the whole organism, we observed an only minimal toxicity after systemic heterozygous loss of Mcl-1 in vivo. We conclude that re-activation of mitochondrial apoptosis by blockade of MCL-1 represents a promising therapeutic strategy to treat T-cell lymphoma.

Inhibition of poly(ADP-ribose) polymerase activity accelerates T-cell lymphomagenesis in p53 deficient mice.

Cells that lack PARP-1 activity are limited in their ability to repair DNA single strand breaks and respond to DNA damage with a strong accumulation of p53 and enhanced rates of apoptotic cell death. We have generated combinatorial mutant mice that both lack p53 and PARP-1 activity due to the expression of a dominant negative PARP-1 allele targeted to T-cells by the lck promoter. Here we report that these double mutant mice develop T-cell lymphoma at a significantly reduced latency period compared to single p53 null mice that are already cancer prone. We demonstrate that the absence of p53 does not only protect T-cells from lck-PARP-DBD transgenic mice from apoptosis but also abrogates the DNA damage induced cell cycle arrest in the G1 phase. T-cells from double mutant mice continue to proliferate after the induction of DNA strand breaks, are limited in their DNA repair capacity and cannot be eliminated by apoptosis. These results indicate that PARP-1 and p53 cooperate in the suppression of tumorigenesis by maintaining genomic integrity after DNA damage through the activation of a G1/S cell cycle checkpoint the initiation of DNA repair and the induction of cell death.

Loss of Fas/Apo-1 receptor accelerates lymphomagenesis in E mu L-MYC transgenic mice but not in animals infected with MoMuLV.

The Fas/Apo-1 receptor is an integral membrane protein that transduces apoptotic signals upon binding to its natural ligand or to specific antibodies. Loss of Fas/Apo-1 receptor leads in (lpr,lpr) mice to a nonmalignant accumulation of abnormal T-cells very probably due to the lack of induction of apoptosis in peripheral T-cells. It has been reported that soluble forms of Fas/Apo-1 receptor that may interfere with apoptotic signaling occur in patients suffering from various forms of lymphoid neoplasms. Therefore, we wished to investigate whether the loss of proper homeostatic regulation through Fas/Apo-1 receptor mediated apoptosis could influence the process of lymphomagenesis. To this end, we performed two experiments (i) we infected (lpr,lpr) animals with Moloney Murine Leukemia Virus (MoMuLV) that causes T-cell lymphoma in mice and (ii) we crossed (lpr,lpr) animals with E mu L-myc transgenic mice that are prone to develop T- and B-cell lymphoma due to deregulated expression of the L-myc transgene by the immunoglobulin enhancer E mu. We find that infection with MoMuLV did not accelerate the formation of lymphoid neoplasms in (lpr,lpr) mice when compared to infected normal animals. However, E mu L-myc/(lpr,lpr) animals that constitutively express the L-myc transgene in the lymphoid lineage clearly show accelerated formation of T- and B-cell lymphoma when compared to normal E mu L-myc transgenics. These data demonstrate that in cooperation with particular oncogenes impairment of Fas/Apo-1 receptor function can indeed affect and modulate the process of tumor formation.

Expression of hpttg proto-oncogene in lymphoid neoplasias.

Pituitary tumor-transforming gene (pttg) is a distinct proto-oncogene which is expressed in certain normal tissues with high proliferation rate and in a variety of tumors. PTTG is the vertebrate analog of yeast securins Pds1 and Cut2 with a key role in the regulation of sister chromatid separation during mitosis. Impairment of PTTG regulated functions is expected to lead to chromosomal instability and aneuploidy. Human pttg (hpttg) is abundantly expressed in Jurkat T lymphoblastic lymphoma cells but not in normal peripheral blood leukocytes. To obtain additional data on the potential role of hpttg in lymphomagenesis we selected 150 cases of lymphoid tumors for the assessment of hpttg expression in tumor tissues. Immunohistochemical studies on formalin-fixed, paraffin-embedded tissues revealed hPTTG in 38.8% of B-cell lymphomas, 70.2% of T-cell lymphomas, and 73.1% of Hodgkin's lymphomas. Among B-cell lymphomas, the most frequently immunostained tumors were plasma cell tumors, diffuse large cell lymphomas, and follicle center cell lymphomas. In Hodgkin's disease, immunoreactivity was mainly noted in Reed-Sternberg cells. In conclusion, the frequent overexpression of hpttg in many histological subtypes of lymphoma suggests the involvement of this proto-oncogene in lymphomagenesis.

Annotated proteome of a human T-cell lymphoma.

As the reliable identification of proteins by tandem mass spectrometry becomes increasingly common, the full characterization of large data sets of proteins remains a difficult challenge. Our goal was to survey the proteome of a human T-cell lymphoma-derived cell line in a single set of experiments and present an automated method for the annotation of lists of proteins. A downstream application of these data includes the identification of novel pathogenetic and candidate diagnostic markers of T-cell lymphoma. Total protein isolated from cytoplasmic, membrane, and nuclear fractions of the SUDHL-1 T-cell lymphoma cell line was resolved by SDS-PAGE, and the entire gel lanes digested and analyzed by tandem mass spectrometry. Acquired data files were searched against the UniProt protein database using the SEQUEST algorithm. Search results for each subcellular fraction were analyzed using INTERACT and ProteinProphet. All protein identifications with an error rate of less than 10% were directly exported into excel and analyzed using GOMiner (NIH/NCI). The Gene ontology molecular function and cell location data were summarized for the identified proteins and results exported as user-interactive directed acyclic graphs. A total of 1105 unique proteins were identified and fully annotated, including numerous proteins that had not been previously characterized in lymphoma, in functional categories such as cell adhesion, migration, signaling, and stress response. This study demonstrates the utility of currently available bioinformatics tools for the robust identification and annotation of large numbers of proteins in a batchwise fashion.

Expression of the human peripheral lymph node homing receptor (LECAM-1) in nodal and gastrointestinal non-Hodgkin's lymphomas.

Lymphocyte migration to lymphoid organs involves tissue-specific homing receptors. A lectin-like surface glycoprotein of 80 kD, the LECAM-1 (LAM-1, Leu-8) antigen, has recently been shown to represent the human equivalent of the mouse peripheral lymph node homing receptor MEL-14. In this study, the expression of LECAM-1 was examined in 116 nodal and 53 gastrointestinal (GI) non-Hogdkin's lymphomas (NHL). This analysis revealed that whereas the majority of nodal lymphomas expressed LECAM-1, this molecule was generally absent on GI lymphomas. This difference was present in each subclass of lymphomas but was most significant among diffuse large-cell lymphomas of the B-lineage (83 versus 23%, p less than 0.0001) and among T-cell lymphomas (89 versus 0%, p less than 0.0001) with a nodal versus GI tract localization. The strong correlation between LECAM-1 expression and the localization of the lymphomas supports the concept that tissue-specific homing receptors, i.e. LECAM-1, play a role in the dissemination of NHL.

Adhesion molecule expression does not influence the leukemic behavior of murine T-cell lymphomas.

Recirculation and homing properties of normal lymphocytes are controlled by interactions with high endothelial venules (HEVs), specialized vessels which mediate the extravasation into lymphoid tissues. The present study was aimed at elucidating whether lymphoma-derived leukemic cell spreading and peripheral lymph node invasion ability are mediated by the recognition mechanisms which physiologically regulate normal lymphocyte trafficking. For this purpose, we tested the HEV-binding ability and the expression of the lymphocyte homing receptor (LHR) for peripheral lymph nodes as well as Pgp-1/CD44, LFA-1 and ICAM-1 adhesion molecules by the highly leukemic cell line NQ22 in comparison with a series of non-leukemic murine T-lymphoma cell lines. Our results indicate that the hematogenous spreading as well as peripheral node invasion of lymphoma-derived leukemic cells may occur independently of LHR expression. In addition, our findings seem to rule out that gross quantitative modifications in LFA-1 or ICAM-1 antigen expression are associated with differential dissemination abilities of transformed lymphoid cells.

Disrupted p53 function as predictor of treatment failure and poor prognosis in B- and T-cell non-Hodgkin's lymphoma.

Mutation of the p53 gene has been associated with treatment failure and poor outcome in various malignancies. It has been suggested that immunohistochemical analysis of p53 and p21Waf1, a downstream target, can be used to screen for p53 gene mutations. We determined the value of immunohistochemical screening for p53 gene mutations as a prognostic marker in a population-based group of B- and T-cell non-Hodgkin's lymphomas (NHLs). On the basis of p53 gene mutation status and immunohistochemically detected p53 and p21Waf1 expression in 34 lymphomas, we established an immunophenotype (delta p53) correlating with p53 gene mutation. The immunohistochemical analysis was extended to encompass 199 lymphomas from a population-based registry and was correlated with clinical parameters. Delta p53 showed 100% concordance with p53 gene mutation and was detected in 42 cases (21%). Multivariate analysis of advanced stage lymphomas showed that delta p53 was independently associated with treatment failure (relative risk, 3.8; P = 0.001). Delta p53 predicted poor survival when analyzing all patients (P = 0.0001), as well as B-cell (P = 0.04) and T-cell NHL (P = 0.000002). In multivariate analysis, delta p53 (relative risk, 2.2; P = 0.001) maintained prognostic significance. The impact on prognosis of delta p53 was highly significant in the low-intermediate-risk group (P = 0.00002). Comparing survival of the aggressive lymphoma patients in this group showed that the 8 delta p53 patients died within 1 year, whereas the median survival of the 28 non-delta p53 patients was 36 months. These results suggest that immunohistochemically assessed p53 status may predict treatment response and outcome in B- and T-cell NHL patients.