Yoshitaka Hosokawa

A novel gene, MALT1 at 18q21, is involved in t(11;18) (q21;q21) found in low-grade B-cell lymphoma of mucosa-associated lymphoid tissue.

The t(11;18) (q21;q21) translocation is a characteristic chromosomal aberration in low-grade B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) type. We previously identified a YAC clone y789F3, which includes the breakpoint at 18q21 in a MALT lymphoma patient. BAC and PAC contigs were constructed on the YAC, and BAC 193f9 was found to encompass the breakpoint region. In the present study, we further narrowed down the breakpoint region at 18q21 in five MALT lymphoma patients by means of FISH and Southern blot analyses using the plasmid contig constructed from BAC 193f9. The breakpoints at 18q21 in three of the five MALT lymphoma patients were found to be clustered approximately within the 20 kb region. By using exon amplification and cDNA library screening, we identified a novel cDNA spanning the breakpoint region that exhibited aberrant mRNA signals in four of the five MALT lymphoma patients. The nucleotide sequence predicted an 813 amino acid protein that shows significant sequence similarity to the CD22β and laminin 5 α3b subunit. We refer to the gene encoding this transcript as MALT1 (Mucosa-Associated Lymphoid Tissue lymphoma translocation gene 1). The alteration of MALT1 by translocation strongly suggests that this gene plays an important role in the pathogenesis of MALT lymphoma.

Molecular cytogenetic delineation of the breakpoint at 18q21.1 in low-grade B-cell lymphoma of mucosa-associated lymphoid tissue.

Extranodal malignant non‐Hodgkin lymphoma of mucosa‐associated lymphoid tissue type (MALT lymphoma) represents a subtype of B‐cell lymphoid malignancies with distinct clinicopathological features and is often associated with a favorable prognosis. Recent cytogenetic studies have revealed that t(11;18)(q21;q21) is a characteristic chromosomal aberration in low‐grade B‐cell MALT‐type lymphoma. In the present study, we employed florescence in situ hybridization analysis using contiguous YAC clones mapped to the 18q21.1 region to identify a YAC clone, y789F3, encompassing the breakpoint of t(11;18)(q21;q21) in a MALT lymphoma. P1 artificial chromosome (PAC) contigs constructed on this YAC clone were used to analyze the breakpoint region. PAC clone 264m4 was observed on normal chromosome 18 and on der(18), and PAC clone 879n10 on normal chromosome 18 and on der(11), confirming that the breakpoint is located between these two PAC clones. We also found that a region of approximately 500 kb between the two PAC clones was deleted. These results indicate that the locus between PAC clones 264m4 and 879n10 at 18q21.1 involved in t(11;18) translocation or associated deletion plays an important role in the development of MALT lymphoma. Genes Chromosomes Cancer 24:315–321, 1999.

Molecular pathogenesis of MALT lymphoma: two signaling pathways underlying the antiapoptotic effect of API2-MALT1 fusion protein.

At least three recurrent chromosomal translocations, t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21), involving the API2-MALT1 fusion protein, BCL10 and MALT1, have been implicated in the pathogenesis of mucosa-associated lymphoid tissue (MALT) lymphoma. Several lines of evidence indicated that both BCL10 and MALT1 are required for nuclear factor kappa B (NF-κB) activation by antigen receptor stimulation in lymphocytes, and API2-MALT1 can bypass this BCL10/MALT1 signaling pathway. Nuclear factor kappa B activation may contribute to antiapoptotic effect through NF-κB-mediated upregulation of apoptotic inhibitor genes. We recently demonstrated that API2-MALT1 can induce transactivation of the API2 gene through NF-κB activation, thus highlighting a positive feedback-loop mechanism of self-activation by upregulating its own expression in t(11;18) MALT lymphomas. We also demonstrated that API2-MALT1 possesses an antiapoptotic effect, in part, through its direct interaction with apoptotic regulators. These findings therefore led us to hypothesize that the antiapoptotic effect by API2-MALT1 may be mediated by its interaction with apoptotic regulators, on the one hand, and by NF-κB-mediated upregulation of apoptotic inhibitor genes on the other. We also found that BCL10 and MALT1 are shuttling between nucleus and cytoplasm, and that MALT1 can regulate the subcellular location of BCL10.

Anti-apoptotic action of API2-MALT1 fusion protein involved in t(11;18)(q21;q21) MALT lymphoma.

At least three distinct chromosomal translocations, t(11;18)(q21;q21), t(1;14)(p22;q32) and t(14;18)(q32;q21) involving the API2 (also known as c-IAP2)-MALT1 fusion protein, BCL10, and MALT1, respectively, have been implicated in the molecular pathogenesis of mucosa associated lymphoid tissue (MALT) lymphoma. Our findings showed that several variants of the API2-MALT1 fusion protein can occur in patients with t(11;18)(q21;q21), and that API2-MALT1 can potently enfance activation of nuclear factor (NF)-κB signaling, which may be relevant to the pathogenesis of MALT lymphomas. We also found that MALT1 is rapidly degraded via the ubiquitin-proteasome pathway, as is the case with API2, but upon the synthesis of fusion, API2-MALT1 becomes stable against this pathway. This stability of API2-MALT1 may thus result in inappropriate nuclear factor (NF)-κB activation, thereby contributing to the pathogenesis of MALT lymphoma. Recent biochemical and genetic studies have clearly shown that BCL10 and MALT1 form a physical and functional complex and are both required for NF-κB activation by antigen receptor stimulation in T and B lymphocytes. It has also been shown that CARMA1, a newly discovered member of the membrane-associated guanylate kinase (MAGUK) families, is critical for antigen receptor-stimulated NF-κB activation. It can be assumed that API2-MALT1 can bypass this normal BCL10/MALT1 cellular signaling pathway linked to NF-κB activation, thereby inducing antigen receptor-independent proliferation of lymphocytes. Furthermore, BCL10/MALT1- and API2-MALT1-induced NF-κB activation may contribute to anti-apoptotic action probably through NF-κB-mediated upregulation of apoptotic inhibitor genes. We recently provided direct evidence that API2-MALT1 indeed exerts anti-apoptotic action, in part, through its direct interaction with apoptotic regulators including Smac. Taken together, these findings prompt us to hypothesize that the anti-apoptotic action of API2-MALT1 may be mediated partly by the direct interaction with apoptotic regulators as well as partly by upregulation of apoptotic inhibitor genes. Further studies can be expected to stimulate research into the development of therapeutic drugs that specifically inhibit the antigen receptor signaling-stimulated NF-κB activation pathway: such molecule targeting drugs should be useful for interfering with inappropriate proliferation of lymphocytes associated with inflammatory and neoplastic disorders.

Stability and subcellular localization of API2-MALT1 chimeric protein involved in t(11;18) (q21;q21) MALT lymphoma.

t(11; 18) (q21; q21) is a chromosomal aberration specific to low-grade mucosa-associated lymphoid tissue (MALT) lymphoma, and generates the chimeric product apoptosis inhibitor 2 (API2)-MALT1, which has been suggested to play an important role in MALT lymphomagenesis. However, little is known about the characteristics of API2, MALT1, and API2-MALT1 proteins. We therefore investigated the subcellular localization and stability of these products. API2 was localized in the nucleus and the cytoplasm, and MALT1 and API2-MALT1 in the cytoplasm only. Western blot analysis showed that the products of API2 and MALT1 were unstable, while the API2-MALT1 product was stable. The API2 deletion mutants at the end of the C-terminal and the MALT1 deletion mutants at the end of the N-terminal were stable compared with the full-length products. These results indicate that the domains responsible for protein instability are located in the end of the C-terminal of API2 and in that of the N-terminal of MALT1, and also that API2-MALT1 became stable because it lacks these domains. It has been suggested that NF-κB activation plays an important role in the tumorigenesis of MALT lymphoma. Our findings further suggest that the stabilized expression of API2-MALT1 products may continuously stimulate the NF-κB activating pathway, thus leading to MALT lymphomagenesis.

A small deletion in the 3′-untranslated region of the cyclin D1/ PRAD1/ bcl-1 oncogene in a patient with chronic lymphocytic leukemia

The cyclin D1/PRAD1 oncogene, a key regulator of the G1 phase of the cell cycle, has been incriminated in the pathogenesis of human neoplasia. Cyclin D1 was also demonstrated to be identical to the long‐sought bcl‐1 oncogene in B‐cell malignancies with the t(11;14)(q13;q32) translocation. We report here a small deletion in the 3′‐untranslated portion of the cyclin D1 gene in leukemia cells of a patient diagnosed with B‐chronic lymphocytic leukemia (CLL), associated with overexpression of the corresponding cyclin D1 mRNA. During a Northern blot survey of B‐cell malignancies, we identified a patient whose CLL cells showed a marked increase in 1.5–1.6 kb cyclin D1 mRNA species. Subsequent Southern blot analysis showed that genomic DNA from the patients cells contained an extra band in the EcoRI digest, suggesting that one allele of the cyclin D1 gene may be altered. Polymerase chain reaction (PCR) analysis of the genomic DNA and direct DNA sequencing clearly disclosed that one allele of the cyclin D1 gene was deleted in the 3′‐untranslated region, which would contribute to an increased stability of its mRNA. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis and direct DNA sequencing revealed that the cyclin D1 mRNA was deleted at the corresponding region. This finding provides further evidence for a critical role of cyclin D1 in the pathogenesis of B‐cell malignancies and highlights a novel mechanism, a small deletion in the 3′‐untranslated region, responsible for deregulation of the cyclin D1 gene in oncogenesis. Int. J. Cancer 76:791–796, 1998.© 1998 Wiley‐Liss, Inc.

Single copies of mutant KRAS and mutant PIK3CA cooperate in immortalized human epithelial cells to induce tumor formation

The selective pressures leading to cancers with mutations in both KRAS and PIK3CA are unclear. Here, we show that somatic cell knockin of both KRAS G12V and oncogenic PIK3CA mutations in human breast epithelial cells results in cooperative activation of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in vitro, and leads to tumor formation in immunocompromised mice. Xenografts from double-knockin cells retain single copies of mutant KRAS and PIK3CA, suggesting that tumor formation does not require increased copy number of either oncogene, and these results were also observed in human colorectal cancer specimens. Mechanistically, the cooperativity between mutant KRAS and PIK3CA is mediated in part by Ras/p110α binding, as inactivating point mutations within the Ras-binding domain of PIK3CA significantly abates pathway signaling. In addition, Pdk1 activation of the downstream effector p90RSK is also increased by the combined presence of mutant KRAS and PIK3CA. These results provide new insights into mutant KRAS function and its role in carcinogenesis.

Combined arsenic trioxide-cisplatin treatment enhances apoptosis in oral squamous cell carcinoma cells

BackgroundOral squamous cell carcinoma (OSCC) accounts for the majority of oral cancers. Despite recent advances in OSCC diagnostics and therapeutics, the overall survival rate still remains low. Here, we assessed the efficacy of a combinatorial arsenic trioxide (ATO) and cisplatin (CDDP) treatment in human OSCC cells.MethodsThe combinatorial effect of ATO/CDDP on the growth and apoptosis of OSCC cell lines HSC-2, HSC-3, and HSC-4 was evaluated using MTT and annexin V assays, respectively. Chou–Talalay analyses were preformed to evaluate the combinatorial effects of ATO/CDDP on the dose-reduction index (DRI). To clarify the mechanism underlying the ATO/CDDP anticancer effect, we also examined the involvement of reactive oxygen species (ROS) in ATO/CDDP-induced apoptosis.ResultsCombination index (CI) analyses revealed that a synergistic interaction of ATO and CDDP elicits a wide range of effects in HSC-2 cells, with CI values ranging from 0.78 to 0.90, where CI < 1 defines synergism. The CI values in HSC-3 and HSC-4 cells ranged from 0.34 to 0.45 and from 0.60 to 0.92, respectively. In addition, ATO/CDDP yielded favorable DRI values ranging from 1.6-fold to 7.71-fold dose reduction. Compared to mono-therapy, ATO/CDDP combinatorial therapy significantly augmented the loss of mitochondrial potential, caspase-3/7 activity and subsequent apoptosis. These changes were all abrogated by the antioxidant N-acetylcysteine.ConclusionsThis study provides the first evidence for a synergistic ATO/CDDP anticancer (apoptotic) activity in OSCC cells with a favorable DRI, thereby highlighting its potential as a combinational therapeutic regime in OSCC.

Cyclin D1/PRAD1/BCL-1 alternative transcript [B] protein product in B-lymphoid malignancies with t(11;14)(q13;q32) translocation

The cyclin‐D1/PRAD1 oncogene, a key regulator of the G1‐phase progression of the cell cycle, has been identified as the long‐sought BCL‐1 oncogene in B‐cell malignancies with t(11;14)(q13;q32) translocation. A novel alternative spliced cyclin‐D1 transcript, called transcript[b], has been identified. The level of the variant transcript[b] was lower than that of the originally reported cyclin‐D1 transcript, called transcript[a], in several human non‐lymphoid cancer cell lines but the endogenous cellular expression of transcript[b] products has not yet been determined. Northern‐blot analysis and reverse‐transcription‐polymerase‐chain‐reaction(RT‐PCR) analysis revealed that transcript[b] mRNA is well expressed in B‐lymphoid cell lines with t(11;14)(q13;q32) translocation and at much lower or undetectable levels in other cells. Western‐blot analysis using a human cyclin‐D1‐specific monoclonal antibody, which can recognize and distinguish the products of transcripts [a] and [b], strongly suggested that the transcript [b] protein is indeed expressed in these B‐cell lines. The present study provides identification of the endogenous cellular expression of the cyclin‐D1‐transcript[b] protein and strongly suggests that this alternative form of cyclin D1 may play a significant role in the molecular pathogenesis of B‐lymphoid malignancies with t(11;14)(q13;q32) translocation. Int. J. Cancer 81:616–619, 1999.

Lipopolysaccharide augments the uptake of oxidized LDL by up-regulating lectin-like oxidized LDL receptor-1 in macrophages

There is a growing body of evidence supporting an intimate association of immune activation with the pathogenesis of cardiovascular diseases, including atherosclerosis. Uptake of oxidized low-density lipoprotein (oxLDL) through scavenging receptors promotes the formation of mature lipid-laden macrophages, which subsequently leads to exacerbation of regional inflammation and atherosclerotic plaque formation. In this study, we first examined changes in the mRNA level of the lectin-like oxLDL receptor-1 (LOX-1) in the mouse macrophage cell line RAW264.7 and the human PMA-induced macrophage cell line THP-1 after LPS stimulation. LPS significantly up-regulated LOX-1 mRNA in RAW264.7 cells; LOX-1 cell-surface protein expression was also increased. Flow cytometry and fluorescence microscopy analyses showed that cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with LPS stimulation. The augmented uptake of Dil-oxLDL was almost completely abrogated by treatment with an anti-LOX-1 antibody. Of note, knockdown of Erk1/2 resulted in a significant reduction of LPS-induced LOX-1 up-regulation. Treatment with U0126, a specific inhibitor of MEK, significantly suppressed LPS-induced expression of LOX-1 at both the mRNA and protein levels. Furthermore, LOX-1 promoter activity was significantly augmented by LPS stimulation; this augmentation was prevented by U0126 treatment. Similar results were also observed in human PMA-induced THP-1 macrophages. Taken together, our results indicate that LPS up-regulates LOX-1, at least in part through activation of the Erk1/2 signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of TLR4-mediated aberrant LOX-1 signaling in the pathogenesis of atherosclerosis.