Background: Proteasome inhibitors (PIs) (e.g., Bortezomib) are key treatments to improve the clinical outcome of multiple myeloma (MM). However, disease relapse from the emergence of PI-resistant clones is the main problem. Insights into the mechanisms underlying PI-resistance will provide novel druggable targets to prevent relapse. It was well-recognized that PIs induce endoplasmic reticulum (ER) stress in MM cells. ER stress is associated with the Calcineurin/nuclear-factor-of-activated-T-cells (NFAT) signaling. Accumulating studies indicated the importance of Calcineurin/NFAT signaling in tumorigenesis and drug resistance. However, the involvement of Calcineurin/NFAT signaling in PI-resistance in MM has not yet been investigated. Aims: To dissect the role of the Calcineurin/NFAT axis in Bortezomib (BTZ) treatment response and explore the therapeutic potential by combining Bortezomib and Calcineurin/NFAT inhibitors in MM. Methods: We studied the expression pattern of PPP3CA and NFATC1 from MM datasets and MM cell lines and their association with BTZ responses in MM. The expression of the four principal NFAT members was detected by expression microarray and their activation status by western blotting in MM cell lines. We identified the most prominent NFAT member and assessed its activation under BTZ treatment. A lentivirus-transduction system was used for overexpressing genes, and siRNA transfection was applied to induce NFATC1 knockdown in a BTZ-resistant cell line ARH77. Flow cytometry and western blotting assessed the combinational effect of Calcineurin/NFAT inhibitors and BTZ on MM cells. Results: The expression level of PPP3CA, which encodes Calcineurin Catalytic subunit A (CnA), was correlated to the poor clinical outcome of recurrent MM patients (MMRF cohort). Calcineurin was highly expressed in ARH77, which is less sensitive to BTZ treatment than other MM cell lines. Among the four NFAT members, we found that NFATC1 and NFATC3 were the most highly expressed members in MM cell lines. However, NFATc1 was the only member suppressed by CsA but activated by the calcium channel agonist Ionomycin in ARH77. Consistent with Calcineurin, NFATc1 was highly expressed in ARH77, with reference to a confirmed NFATc1-positive cell line Raji and the nuclear-NFATc1 overexpressing HEK-293T. Additionally, BTZ activated CnA and NFATc1 time-dependently in ARH77 but failed to activate CnA and NFATc1 in the BTZ-sensitive cell line NCI-H929. Immunofluorescence showed that BTZ triggered the nuclear translocation of NFATc1. From GEO datasets, NFATC1 was overexpressed in patients less responsive to BTZ, but was unchanged in patients subjected to conventional therapies. The Calcineurin/NFATc1 was thus a potential mechanism of BTZ-resistance in MM. Depleting NFATc1 enhanced BTZ-induced DNA damage-related apoptosis in ARH77. The combinational treatment of Calcineurin/NFAT inhibitors (CsA and FK506) and BTZ decreased the nuclear/cytosolic ratio of NFATc1 and promoted apoptosis in two MM cell lines compared to the treatment of BTZ alone. Image:Summary/Conclusion: The activation of the Calcineurin/NFATc1 axis is an important mechanism that contributes to BTZ-resistance in MM. Targeting this axis confers translational potential for improving BTZ-based therapies.
Originally referred to as 'lymphoepithelioma' undifferentiated and poorly differentiated nasopharyngeal carcinoma (NPC) tissues showed intense lymphoinfiltration. In a study of cryosections from 15 NPC tissues, we found that infiltrating lymphoid elements were comprised predominantly of lymphocytes, but plasma cells, follicular dendritic cells, and eosinophils were also commonly seen. Subpopulations of lymphocytes having the same phenotypes tend to aggregate, forming clusters or secondary follicles in stromatous tissues. The tumor areas were mainly infiltrated by T cells. Tumor cells and/or apparently normal epithelium in the paratumorous areas frequently expressed CD21, CD23, CD40 and a B lymphocytes carcinoma cross-reacting antigen (BLCa), all of which are involved in B cell activation and proliferation. CD21 and BLCa were strongly expressed near the surface of both squamous and columnar epithelium by those epithelial cells which are at advanced stage of differentiation, while CD40 was expressed by epithelial cells at earlier stages of differentiation located at or near the basement membrane. CD23 was mainly expressed by columnar cells and basal cells underlying squamous epithelium, but not, or weakly so, by flattened squamous cells or reserve cells underlying columnar epithelium. The large majority of tumor cells expressed CD40 and BLCa. A substantial-proportion of them also expressed CD23, but the tumor cells were not reactive for CD21. Despite eosinophilic infiltration, IL-6 was not detected in tumor tissues. IL-I was, however, detected in abundance in the cytoplasm of follicular dendritic-like cells and in the intercellular spaces in tumor areas and surrounding stromatous tissues. The immunobiology of NPC is discussed in the light of these observations.
Abstract We studied the distribution of the EBV genome in tumour biopsies obtained from 42 patients with poorly differentiated or undifferentiated nasopharyngeal carcinoma (NPC) and 3 patients with well‐differentiated NPC. Six carcinoma in situ (CIS) foci were seen in 5 tumour specimens. By in‐situ hybridization, multiple copies of the EBV genome were detected in some of the tumour cells in 3 CIS lesions involving the full thickness of the mucosal epithelium, but without microinvasion, while the viral genome was present in the majority of the tumour cells contained in another 3 CIS lesions with microinvasion. In agreement with previous findings, poorly differentiated and undifferentiated carcinomas regularly carried the viral genome, the number of copies of which was similar to that seen in CIS, while some, but not all, of the tumour cells of the welldifferentiated histological type carried the virus. The viral genome was otherwise rarely detected in other areas of the mucosal epithelium and, where present, the viral carriage was confined to a few epithelial cells, in which the viral genome contents were markedly lower than in tumour cells. These results suggest that EBV may first become associated with NPC at an early stage of the disease shortly after the tumour has been initiated.
To study the outcome of children with acute lymphoblastic leukaemia who were treated using a protocol including one or two delayed intensifications.Prospective single-arm multicentre study.Five designated children cancer units of the Hospital Authority of Hong Kong.Children aged between 1 and 17.9 years with newly diagnosed acute lymphoblastic leukaemia seen from November 1997 to December 2002.Chemotherapy was modified from a German Berlin-Frankfurt-Muenster 95 (BFM95) protocol that included a delayed intensification similar to the induction phase repeated 5 months after diagnosis. High-risk patients were given double delayed intensification.Overall survival and event-free survival of the whole group and the three risk groups (standard-, intermediate-, and high-risk groups), and comparison with historical controls.A total of 171 patients were recruited with a median age at diagnosis of 5.57 years (range, 1.15-17.85 years). The induction remission rate was 95.3% and non-leukaemia mortality during remission was 2.3%. At 4 years, the relapse rate of this (HKALL97) study was significantly lower than that of the HKALL93 study (15.7 vs 37.3%; P<0.001). The 4-year overall survival of HKALL97 and HKALL93 studies were 86.5% and 81.8%, respectively (P=0.51). The 4-year event-free survival for HKALL97 and HKALL93 studies were 79% and 65%, respectively (P=0.007). Nonetheless the difference of event-free survival was most remarkable in the intermediate-risk group: 75.6% and 53.1% for HKALL97 and HKALL93 studies, respectively (P=0.06).A more intensive delayed consolidation phase improved the outcome for children with acute lymphoblastic leukaemia by reducing relapses at 4 years. The early treatment complications were manageable and non-leukaemia mortality during remission remained low.
Fiberoptic endoscopic examination and biopsy of the nasopharynx was done in 130 patients as a prospective study. They all had elevated titers of antibodies against the viral capsid antigen of Epstein-Barr virus but no symptoms or signs of nasopharyngeal carcinoma. Each underwent a biopsy from six fixed sites in the nasopharynx. Of the 780 biopsy specimens taken from seven patients, 11 showed the presence of nasopharyngeal carcinoma. Techniques to improve the yields of such biopsies with fiberoptic endoscopy are discussed. The presence of tumor was unrelated to the macroscopic endoscopic findings. The highest incidence of subclinical tumor is in the pharyngeal recess.
Aim: Telomerase activity was studied in invasive uterine cervical carcinoma to assess whether it was activated during cervical malignant transformation and to look for a possible association with human papillomavirus (HPV) infection in a set of Malaysian patients.Methods: Histologically confirmed invasive cervical carcinoma and benign cervices were assayed for telomerase activity using a commercial telomerase polymerase chain reaction (PCR) enzyme linked immunosorbent assay kit.The same cases were subjected to PCR detection of HPV using type specific (HPV types 6b, 11, 16, and 18) followed by L1 open reading frame (ORF) consensus primers.Results: HPV was detected in 18 (13 HPV-16, one HPV-6b, four only L1 ORF) of 20 invasive cervical carcinoma and one (only L1 ORF) of 19 benign cervices.Raised telomerase activity (A 450 nm > 0.215) was detected in 11 cervical carcinomas, with A 450 nm ranging between 0.238 and 21.790 (mean, 3.952) in positive squamous carcinomas, whereas A 450 nm was only 0.222 in the one positive adenosquamous carcinoma.Five of 11 cervical carcinomas in stage I, three of six in stage II, both in stage III, and the only case in stage IV showed telomerase activation.Increased telomerase activity was noted in five of the 12 lymph node negative, five of the seven lymph node status unknown cases, and the one case with presumed lymph node metastasis.Ten of 18 HPV positive and one of two HPV negative cervical carcinomas showed telomerase upregulation.Conclusions: Telomerase is activated in invasive cervical carcinoma.Although larger studies are needed, there seems to be no clear association between telomerase upregulation and HPV status, although there is a suggestion of increased telomerase activity in squamous carcinomas and late stage disease.. . . . . . . . .
The genome of naturally occurring Epstein-Barr virus contains either two or three copies of a 29-bp tandem repeat sequence in the first intron of the BZLF gene. These genotypes differ markedly in their distribution between blood and epithelial tissues, presumably because they have adapted to separate life cycles in these sites. The genotype prevalent in the blood also appears to be better able to transform B lymphocytes.
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