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As compared to the studies with hot water extracts of plants, those with alkaline extracts were limited. Both alkaline and hot water extracts from green tea leaf, oolong tea leaf and orange flower were compared for their biological activities. Plant materials were successively extracted first with hot-water and then alkaline solution, or extracted directly with alkaline solution. Viable cell number of HIV-infected and UV-irradiated cells was determined by MTT method. Antibacterial activity against Porphyromonas gingivalis 381 was determined by turbidity assay. Cytochrome P-450 (CYP)3A4 activity was measured by β-hydroxylation of testosterone using human recombinant CYP3A4 (Figure 5). Radical intensity of superoxide and hydroxyl radical was determined by ESR spectroscopy. Alkaline extraction recovered twice as much as dried materials as compared with water extraction. Water extracts showed higher anti-bacterial, CYP3A4 inhibitory and superoxide scavenging activities, whereas alkaline extract showed higher anti-HIV and hydroxyl radical scavenging activity. Both water and alkaline extracts showed comparable anti-UV activity. The present study suggests the usefulness of alkaline extraction for the efficient utilization of the natural resources.

The association of Epstein-Barr virus (EBV) with smooth-muscle tumors was recently reported in the setting of acquired immunodeficiency syndrome (AIDS) and post-transplantation. We report a case of an EBV-associated smooth-muscle tumor arising in a post-transplant (PT) patient who previously was treated successfully for two EBV-associated PT large-cell lymphomas. A 4-year-old girl required cardiac transplantation for dilated cardiomyopathy when she was aged 23 months. Her PT regimen included cyclosporine, azothiaprine, and diltiazem. At 16 months PT, she presented with anemia, guaiac-positive stools, and an abdominal mass diagnosed as diffuse large-cell lymphoma of B-cell phenotype. Immunosuppressive therapy was reduced, and interferon and i.v. immunoglobulin were initiated. She rapidly developed signs of rejection, and a cardiac biopsy was performed, revealing grade IIIB rejection. Subsequently, immunosuppressive therapy increased. At 23 months PT, a biopsy was done of a large pelvic mass that was diagnosed as immunoblastic large-cell lymphoma. After treatment with chemotherapy and retinoic acid, the size of the mass markedly decreased. Follow-up computed tomography scan revealed multiple liver nodules. A needle biopsy of the liver showed a smooth-muscle tumor of indeterminate grade. Both the lymphomas and the smooth-muscle tumor contained EBV within > 95% of tumor cells by Epstein-Barr (EBER1) in situ hybridization, were of strain type A by Epstein-Barr nuclear antigen-2 (EBNA-2) polymerase chain reaction (PCR) and contained an identical 30 base-pair deletion (amino acids 346-355) of the latent membrane protein (LMP)-1 oncogene by PCR analysis. Notably, the initial large-cell lymphoma and the subsequent immunoblastic lymphoma each contained a unique p53 mutation, suggesting that they were distinct. These data suggest that the same virus contributed to the pathogenesis of both the malignant lymphomas and the smooth-muscle tumor.

Multiple lymphomatous polyposis (MLP), characterized by multiple polyps involving long segments of the gastrointestinal (GI) tract, is believed to represent GI involvement by mantle cell lymphoma (MCL), primarily based on its histologic and immunophenotypic similarities with MCL. However, rearrangement of the bcl -l locus, the molecular lesion characteristic of MCL, has not been investigated in this group of patients. The authors evaluated the morphologic, immunophenotypic,and molecular features of 18 cases of MLP and 8 B-cell lymphomas involving the GI tract (including 6 MALT lymphomas). All MLP cases presented with GI disease, and were histologically similar to MCL. DNA extracted from formalin-fixed, paraffin-embedded tissue was analyzed for evidence of bcl -l rearrangement by PCR, using chromosome 11 specific and consensus JH primers. Amplifiable DNA was obtained in 24 of 26 cases (16 of 18 MLP cases and 8 of 8 controls), bcl -l re-arrangement was detected in 6 of 16 cases (38%), subsequently confirmed by sequencing of the breakpoint region, and in 0 of 8 controls. Immunostaining for cyclin Dl was positive in 14 of 18MLPs, including the 6 bcl -l rearranged cases and negative in 6 of 6 evaluable controls. The detection of bcl -l rearrangement and cyclin Dl expression in cases of MLP supports the view that MLP represents primary MCL of the GI tract. These techniques may also be helpful in differentiating MLP from other GI lymphomas, particularly low grade lymphomas of MALT, when only small routinely fixed endoscopic biopsies are available.

The t(2;5)(p23;q35) translocation is associated with a high percentage of anaplastic large-cell lymphomas (ALCL) of T- or null-cell phenotype. This translocation was recently cloned and results in the fusion of the nucleophosmin gene (NPM) on chromosome 5q35 to a novel tyrosine kinase- encoding gene designated anaplastic lymphoma kinase (ALK) on chromosome 2p23. Using a sensitive and specific reverse transcription-polymerase chain reaction (RT-PCR) assay to detect the NPM/ALK fusion transcript, we assessed the involvement of NPM/ALK in a series of histologically and immunohistochemically confirmed ALCL, in non-ALCL aggressive non- Hodgkin's lymphomas of T-cell phenotype, and in Hodgkin's disease (HD) to better define the morphologic spectrum of disease associated with this translocation. Twenty-four cases of ALCL were selected on the basis of CD30 positivity and histologic features. Seventeen cases presented as classical nodal and extranodal disease, four cases presented as primary cutaneous disease, and three were associated with human immunodeficiency virus (HIV) infection. As ALCL may show overlapping histology with both HD and other aggressive non-Hodgkin's lymphomas, particularly of T-cell phenotype (T-NHL), we also studied 34 cases of HD and 19 of T-NHL. NPM/ALK chimeric transcripts of identical size were detected in 11 of the 24 (46%) cases of ALCL. NPM/ALK fusion transcripts were found in 11 of 17 (65%) classical ALCL cases but were not detected in the four primary cutaneous cases of ALCL or in the three HIV-related ALCL cases. In addition, NPM/ALK transcripts were not detected in any of the 34 cases of HD or in the 19 cases of T-NHL. These data indicate that NPM/ALK fusion transcripts occur in a high percentage of classical nodal ALCL (65%). In addition, these data strongly suggest that ALCL, as defined in this study, is not pathogenetically related to either HD disease or the majority of other types of aggressive T-NHL. This is a US government work. There are no restrictions on its use.

Measurement of P-glycoprotein and the gene that encodes it, mdr-1, is an important tool for assessing the impact of multidrug resistance in clinical cancer. We evaluated mdr-1 expression by a quantitative polymerase chain reaction (PCR) assay in 78 biopsy samples from 48 patients with refractory lymphoma enrolled on a trial of infusional chemotherapy (EPOCH) in which R-verapamil was added as an antagonist of P-glycoprotein in a subset of patients whose tumors were unresponsive to treatment. Expression of mdr-1 was detectable in all biopsies at the time of enrollment on study, and a fourfold or greater increase in mdr- 1 expression was noted in 42% of patients at the time of treatment failure. Expression of mdr-1 was also detectable in biopsies from patients at the time of diagnosis of lymphoma. An endogenous control gene, beta 2-microglobulin, was quantitated for normalization of the mdr-1 values. The use of beta 2-microglobulin expression for normalization was validated in a subset of samples by comparing Northern blots detecting beta 2-microglobulin, beta actin, and GAPDH gene expression. Immunoblot analysis suggested that no major discrepancy was present between mRNA expression and protein level. Immunophenotyping of lymphomatous lymph nodes showed that infiltration of tumor cells ranged from 8% to 95% and of normal T cells from 1% to 83%. Expression of mdr-1 in normal T cells and monocytes was also shown to be low. The mdr-1 levels in patient samples were independent of T- cell contamination, suggesting that the presence of normal cells has at best a small impact on mdr-1 measurements. Expression of mdr-1 in lymphoma can be quantitated by PCR, and wide variations in expression can be observed. Increased expression in patients with refractory disease supports an important role for Pgp in drug resistance in lymphoma. These studies will aid in the design and interpretation of clinical trials in lymphoma.

The LAZ3 gene encodes a novel zinc-finger protein that shares homology with several Drosophila transcription factors. This gene was identified by its disruption in translocations involving chromosome 3q27 in diffuse large-cell lymphomas. To assess the frequency and role of this gene's involvement in lymphomagenesis and tumor progression, we examined a series of 170 cases of non-Hodgkin's lymphomas of B-cell lineage for LAZ3 gene rearrangement, expression, and mutation. The cases included 35 de novo diffuse aggressive lymphomas (DAL; 19 large-cell, 4 mixed-cell, and 12 large-cell immunoblastic), 52 transformed aggressive lymphomas derived from follicular lymphomas (TFL), 42 indolent follicular lymphomas (FL), 14 mantle cell lymphomas (MCL), and 27 small noncleaved cell lymphomas (SNCL). LAZ3 rearrangements were found in 10 DAL (28.6%), 9 TFL (17.3%), and 6 FL (14.3%), but not in any of the SNCL or MCL. LAZ3 rearrangement was not exclusive of bcl-2 rearrangement. Most rearrangement breakpoints mapped to a 10-kb BamHI-Xba I fragment located 5′ to the LAZ3 coding sequence, consistent with previously reported breakpoint locations. Northern analysis of both rearranged and nonrearranged B-cell lymphoma cases showed similar levels of a transcript of approximately 3.8 kb, indicating that LAZ3 is broadly expressed in B-cell tumors and is not confined to rearranged cases. To investigate whether mutation of the LAZ3 gene might contribute to a potential role for this gene in lymphomagenesis, we screened the coding sequences of 13 rearranged cases, 6 nonrearranged cases, and 13 hematopoietic tumor cell lines. Although three probable polymorphisms were identified, mutations were detected in only 2 rearranged cases. Only 1 of these resulted in an amino acid substitution. Two cell lines (SU-DHL4 and Molt-4) also contained mutations; only one resulted in an amino acid substitution. We conclude (1) that LAZ3 rearrangements occur in a significant fraction of de novo DAL as well as in a smaller subset of indolent and transformed follicular lymphomas; (2) that LAZ3 message is expressed in both rearranged and nonrearranged B-cell lymphomas; and (3) that mutation of the LAZ3 gene does not contribute to its putative oncogenic role in most 3q27 translocated B-cell lymphomas.

Translocation of the Myc gene on chromosome 8q24 to an immunoglobulin gene segment on chromosomes 14q32, 22q11 or 2p12 is believed to be the central event in the pathogenesis of Burkitt’s lymphoma (Dalla-Favera et al. 1982). The molecular heterogeneity of Myc translocations in Burkitt’s lymphoma, suggests that there may be several mechanisms that account for the resulting deregulation of Myc gene transcription, and the consequent abnormalities in cell growth regulation that lead to neoplasia. For example, in many cases of Burkitt’s lymphoma, the transcriptional enhancer of the immunoglobulin heavy chain gene, Eμ, is located on the same chromosome as the translocated Myc gene, suggesting that it may be responsible for activating Myc transcription (Taub et al. 1982). In other cases, however, Eμ and Myc are located on different chromosomes (Rabbitts et al. 1983), and other mechanisms must operate. In sporadic Burkitt’s lymphomas the breakpoints on chromosome 8q24 frequently occur in intron 1 of the Myc gene, separating the oncogene’s regulatory elements from its coding sequences (Pelicci et al. 1986). This suggests that loss of normal regulatory elements may also play a role in abnormal regulation of the translocated oncogene (Taub et al. 1984; Bentley and Groudine 1988; Hann et al. 1988).

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at chromosome 14q32 represent a common mechanism of oncogene activation in lymphoid malignancies. In multiple myeloma (MM), the most consistent chromosomal abnormality is the 14q+ marker, which originates in one third of cases through a t(11; 14)(q13; q32) chromosomal translocation; in the remaining cases, the identity of the partner chromosomes has not been well established. We used a Southern blot approach based on the linkage analysis of the joining (J) and the constant (C) mu, alpha, and gamma regions to detect cases bearing IGH switch-mediated chromosomal translocations. We evaluated DNA of 88 nonkaryotyped patients with MM (78 cases) or plasma cell leukemia (PCL) (10 cases) and found the presence of "illegitimate" rearranged IGH fragments (no comigration between the J and C regions) in 21 cases. To confirm this analysis, we cloned the illegitimate rearranged fragments from three samples, and the molecular and fluorescent in situ hybridization (FISH) analyses indicated the presence of chromosomal translocations juxtaposing a switch IGH region to sequences from chromosomes 11q13 (one PCL case) or 4p16.3 (two MM cases). Interestingly, the breakpoints on 4p16.3 occurred about 14 kb apart in a genomic region located approximately 50 kb centromeric to the fibroblast growth-factor receptor 3 (FGFR3) gene. Moreover, Southern blot analysis using 4p16.3 genomic probes detected a rearrangement in an additional MM tumor. FISH analysis of the MM-derived KMS-11 cell line, reported to be associated with a t(4; 14)(p16.3; q32), showed that the FGFR3 gene was translocated on 14q32. High levels of FGFR3 mRNA expression were observed in the cloned MM tumors and KMS-11 cell line, but not in the cases that were apparently negative for this lesion. Furthermore, a point mutation at codon 373 in the transmembrane domain of the FGFR3 gene resulting in an amino acid substitution (Tyr --Cys) was detected in the KMS-11 cell line. These findings indicate that the t(4; 14)(p16.3; q32) represents a novel, recurrent chromosomal translocation in MM, and suggest that the FGFR3 gene may be the target of this abnormality and thus contribute to tumorigenesis in MM.