Predictors of lung injury during durvalumab maintenance therapy following concurrent chemoradiotherapy in unresectable locally advanced non-small cell lung carcinoma.

BACKGROUND: Based on the results of the PACIFIC trial, maintenance with durvalumab has emerged as the standard treatment following concurrent chemoradiotherapy in patients with unresectable locally advanced non-small cell lung carcinoma (NSCLC). However, adverse events attributed to durvalumab, especially lung injuries, including immune-related adverse events, and radiation pneumonitis, are concerning. This study retrospectively investigated the factors related to lung injury in patients receiving the PACIFIC regimen. METHODS: Patients with unresectable locally advanced NSCLC who received durvalumab maintenance therapy following concurrent chemoradiotherapy at Yokohama City University Medical Centre between July 2018 and March 2022 were included. Clinical data, volume of normal lung receiving 20 or 5 Gy or more (V20 or V5), planning target volume (PTV), and relative lung parenchyma volume in emphysematous lung receiving 20 or 5 Gy or more (RLPV20 or 5; V20 or V5/100-percentage of low-attenuation volume) were evaluated. RESULTS: Performance status (PS), V20, V5, PTV, RLPV20, and RLPV5 were significantly higher in the lung injury group in the univariate analysis. Furthermore, RLPV20 was the most significant factor in the lung injury group in the multivariate analysis comprising PS, PTV, V20, and RLPV20. CONCLUSION: RLPV20 and RLPV5 are useful in estimating lung inflammation. RLPV20 could be considered the most reliable risk factor for maintenance therapy with durvalumab following concurrent chemoradiotherapy in patients with unresectable locally advanced NSCLC.

Thymic atypical carcinoid tumors with elevated mitotic counts in a patient with multiple endocrine neoplasia: A case report.

Thymic neuroendocrine tumors associated with multiple endocrine neoplasia are only defined as carcinoid and are not associated with large-cell neuroendocrine carcinoma (LCNEC). We report the case of a multiple endocrine neoplasia type 1 patient with atypical carcinoid tumors with elevated mitotic counts (AC-h), an intermediate condition between carcinoid and LCNEC. A 27-year-old man underwent surgery for an anterior mediastinal mass and was diagnosed with thymic LCNEC. Fifteen years later, a mass appeared at the same site, which was determined to be a postoperative recurrence based on the pathological results of a needle biopsy and the clinical course. The patient's disease remained stable for 10 months on anti-programmed death-ligand 1 antibody and platinum-containing chemotherapy. The needle biopsy specimen was submitted for next-generation sequencing, which revealed a MEN1 gene mutation, and after further examination, a diagnosis of multiple endocrine neoplasia type 1 was made. A re-examination of the surgical specimen from 15 years prior showed that it corresponded to AC-h. Although thymic AC-h is classified as thymic LCNEC according to the current definition, our data suggests that a search for multiple endocrine neoplasia is warranted in such patients.

Primary tracheal small-cell carcinoma detected 11 months after surgery for pulmonary large-cell neuroendocrine carcinoma: A case report.

Primary tracheal small-cell carcinoma is rare, and is often treated using small-cell lung cancer guidelines given that no standard treatment has been established for it. We report a patient in whom nodules appeared in the trachea and left main bronchus 11 months after surgery for pulmonary large-cell neuroendocrine carcinoma; a biopsy revealed small-cell carcinoma. Given the absence of malignant lesions elsewhere in the body, the lesions were diagnosed as primary tracheal small-cell carcinoma. Respiratory failure progressed rapidly owing to airway stenosis caused by the growing lesion, and the patient required nasal high-flow therapy. However, the lesions shrank a few days after commencing first-line chemotherapy, and his respiratory failure resolved. Accelerated hyperfractionated radiotherapy was administered in conjunction with the third course of chemotherapy, and the patient ultimately achieved a complete response. Although the lesions were initially suspected of being postoperative recurrence of pulmonary large-cell neuroendocrine carcinoma, the fact that the biopsy revealed them to be primary tracheal small-cell carcinoma indicates that intra-airway nodules that appear after lung cancer surgery may possibly be primary tracheal tumors.

Retrospective analysis of independent predictors of progression-free survival in patients with EGFR mutation-positive advanced non-small cell lung cancer receiving first-line osimertinib.

BACKGROUND: Clinically measurable factors affecting the progression-free survival (PFS) of patients receiving osimertinib as first-line therapy for epidermal growth factor receptor (EGFR) mutation-positive advanced non-small cell lung cancer (NSCLC) have not yet been established. METHODS: We retrospectively reviewed the medical records of 61 patients treated with osimertinib as primary therapy for EGFR mutation-positive advanced NSCLC at Yokohama City University Medical Center between August 2018 and March 2022. Our objective was to identify the independent predictors of PFS. RESULTS: The median age of participants was 74 years. Overall, 73.8% had good (0-1) Eastern Cooperative Oncology Group performance status (PS), and 98.4% had histology of adenocarcinoma. The EGFR mutation was exon19 deletion in 52.5% and exon21 L858R in 44.3% of patients. Programmed death-ligand 1 tumor proportion score >50% was observed in 21.3% and liver metastasis in 9.9% of patients. Median PFS was 19.5 months (95% confidence interval [CI]: 10.6-31.6), and overall survival was not reached. The objective response rate was 68.9%, and disease control rate was 93.4%. Multivariate analysis showed that poor PS (2-4) negatively impacted PFS (hazard ratio, 3.79; 95% CI: 1.46-9.87; p = 0.006). Median PFS in the good PS and poor PS groups was 20.4 months (95% CI: 12.4-not evaluable) and 7.2 months (95% CI: 7.2-19.5), respectively. Interstitial lung disease of all grades and grade 3 was observed as an adverse event in 6.6 and 4.9% of patients, respectively. CONCLUSION: Poor PS was associated with poor prognosis in patients with EGFR mutation-positive advanced NSCLC treated with osimertinib as first-line therapy.

Combination of Pembrolizumab With Platinum-containing Chemotherapy for Pulmonary Enteric Adenocarcinoma.

Background/Aim: Pulmonary enteric adeno-carcinoma (PEAC) is a rare type of non-small cell lung cancer (NSCLC), for which no established standard treatment exists. Combination therapy with the anti-programmed cell death protein 1 antibody pembrolizumab and platinum-containing chemotherapy is the standard treatment for NSCLC patients, but its effectiveness in PEAC is uncertain. Case Report: We present a 68-year-old man with chemotherapy-naïve advanced PEAC who responded to a combination of pembrolizumab and platinum-containing chemotherapy. Conclusion: The number of PEAC cases is small, and no clinical trials have been conducted to determine an optimal chemotherapy regimen. In this case, we showed that pembrolizumab combined with platinum-containing chemotherapy might effectively treat PEAC.

Genetic Diversity of African Trypanosomes in Tsetse Flies and Cattle From the Kafue Ecosystem.

We clarified the genetic diversity of Trypanosoma spp. within the Kafue ecosystem, using PCR targeting the internal transcribed spacer 1 and the cathepsin L-like cysteine protease (CatL) sequences. The overall prevalence of Trypanosoma spp. in cattle and tsetse flies was 12.65 and 26.85%, respectively. Cattle positive for Trypanosoma vivax had a significantly lower packed cell volume, suggesting that T. vivax is the dominant Trypanosoma spp. causing anemia in this area. Among the 12 operational taxonomic units (OTUs) of T. vivax CatL sequences detected, one was from a known T. vivax lineage, two OTUs were from known T. vivax-like lineages, and nine OTUs were considered novel T. vivax-like lineages. These findings support previous reports that indicated the extensive diversity of T. vivax-like lineages. The findings also indicate that combining CatL PCR with next generation sequencing is useful in assessing Trypanosoma spp. diversity, especially for T. vivax and T. vivax-like lineages. In addition, the 5.42% prevalence of Trypanosoma brucei rhodesiense found in cattle raises concern in the community and requires careful monitoring of human African trypanosomiasis.

Targeting of RNA Polymerase II by a nuclear Legionella pneumophila Dot/Icm effector SnpL.

The intracellular pathogen Legionella pneumophila influences numerous eukaryotic cellular processes through the Dot/Icm-dependent translocation of more than 300 effector proteins into the host cell. Although many translocated effectors localise to the Legionella replicative vacuole, other effectors can affect remote intracellular sites. Following infection, a subset of effector proteins localises to the nucleus where they subvert host cell transcriptional responses to infection. Here, we identified Lpw27461 (Lpp2587), Lpg2519 as a new nuclear-localised effector that we have termed SnpL. Upon ectopic expression or during L. pneumophila infection, SnpL showed strong nuclear localisation by immunofluorescence microscopy but was excluded from nucleoli. Using immunoprecipitation and mass spectrometry, we determined the host-binding partner of SnpL as the eukaryotic transcription elongation factor, Suppressor of Ty5 (SUPT5H)/Spt5. SUPT5H is an evolutionarily conserved component of the DRB sensitivity-inducing factor complex that regulates RNA Polymerase II dependent mRNA processing and transcription elongation. Protein interaction studies showed that SnpL bound to the central Kyprides, Ouzounis, Woese motif region of SUPT5H. Ectopic expression of SnpL led to massive upregulation of host gene expression and macrophage cell death. The activity of SnpL further highlights the ability of L. pneumophila to control fundamental eukaryotic processes such as transcription that, in the case of SnpL, leads to global upregulation of host gene expression.

Interactive transcriptome analysis of malaria patients and infecting Plasmodium falciparum.

To understand the molecular mechanisms of parasitism in vivo, it is essential to elucidate how the transcriptomes of the human hosts and the infecting parasites affect one another. Here we report the RNA-seq analysis of 116 Indonesian patients infected with the malaria parasite Plasmodium falciparum (Pf). We extracted RNAs from their peripheral blood as a mixture of host and parasite transcripts and mapped the RNA-seq tags to the human and Pf reference genomes to separate the respective tags. We were thus able to simultaneously analyze expression patterns in both humans and parasites. We identified human and parasite genes and pathways that correlated with various clinical data, which may serve as primary targets for drug developments. Of particular importance, we revealed characteristic expression changes in the human innate immune response pathway genes including TLR2 and TICAM2 that correlated with the severity of the malaria infection. We also found a group of transcription regulatory factors, JUND, for example, and signaling molecules, TNFAIP3, for example, that were strongly correlated in the expression patterns of humans and parasites. We also identified several genetic variations in important anti-malaria drug resistance-related genes. Furthermore, we identified the genetic variations which are potentially associated with severe malaria symptoms both in humans and parasites. The newly generated data should collectively lay a unique foundation for understanding variable behaviors of the field malaria parasites, which are far more complex than those observed under laboratory conditions.

MDM2 regulates a novel form of incomplete neoplastic transformation of Theileria parva infected lymphocytes.

Our efforts are concerned with identifying features of incomplete malignant transformation caused by non viral pathogens. Theileria parva (T. parva) is a tick-transmitted protozoan parasite that can cause a fatal lymphoproliferative disease in cattle. The T. parva-infected lymphocytes display a transformed phenotype and proliferate in culture media like the other tumor cells, however those cells will return to normal after antiprotozoal treatment reflecting the incomplete nature of transformation. To identify signaling pathways involved in this form of transformation of T. parva-infected cells, we screened a library of anticancer compounds. Among these, TIBC, a specific inhibitor of MDM2, markedly inhibited proliferation of T. parva-infected lymphocytes and promoted apoptosis. Therefore we analyzed MDM2 function in T. parva-infected cells. Several T. parva-infected cell lines showed increased expression level of MDM2 with alternatively spliced isoforms compared to the lymphoma cells or ConA blasts. In addition, buparvaquone affected MDM2 expression in T. parva transformed cells. Moreover, p53 protein accumulation and function were impaired in T. parva-infected cells after cisplatin induced DNA damage despite the increased p53 transcription level. Finally, the treatment of T. parva-infected cells with boronic-chalcone derivatives TIBC restored p53 protein accumulation and induced Bax expression. These results suggest that the overexpression of MDM2 is closely linked to the inhibition of p53-dependent apoptosis of T. parva-infected lymphocytes. Aberrant expression of host lymphocyte MDM2 induced by cytoplasmic existence of T. parva, directly and/or indirectly, is associated with aspects of this type of transformation of T. parva-infected lymphocytes. This form of transformation shares features of oncogene induced malignant phenotype acquisition.

A pilot study on developing mucosal vaccine against alveolar echinococcosis (AE) using recombinant tetraspanin 3: Vaccine efficacy and immunology.

BACKGROUND: We have previously evaluated the vaccine efficacies of seven tetraspanins of Echinococcus multilocularis (Em-TSP1-7) against alveolar echinococcosis (AE) by subcutaneous (s.c.) administration with Freund's adjuvant. Over 85% of liver cyst lesion number reductions (CLNR) were achieved by recombinant Em-TSP1 (rEm-TSP1) and -TSP3 (rEm-TSP3). However, to develop an efficient and safe human vaccine, the efficacy of TSP mucosal vaccines must be thoroughly evaluated. METHODOLOGY/PRINCIPAL FINDINGS: rEm-TSP1 and -TSP3 along with nontoxic CpG ODN (CpG oligodeoxynucleotides) adjuvant were intranasally (i.n.) immunized to BALB/c mice and their vaccine efficacies were evaluated by counting liver CLNR (experiment I). 37.1% (p < 0.05) and 62.1% (p < 0.001) of CLNR were achieved by these two proteins, respectively. To study the protection-associated immune responses induced by rEm-TSP3 via different immunization routes (i.n. administration with CpG or s.c. immunization with Freund's adjuvant), the systemic and mucosal antibody responses were detected by ELISA (experiment II). S.c. and i.n. administration of rEm-TSP3 achieved 81.9% (p < 0.001) and 62.8% (p < 0.01) CLNR in the liver, respectively. Both the immunization routes evoked strong serum IgG, IgG1 and IgG2α responses; i.n. immunization induced significantly higher IgA responses in nasal cavity and intestine compared with s.c. immunization (p < 0.001). Both immunization routes induced extremely strong liver IgA antibody responses (p < 0.001). The Th1 and Th2 cell responses were assessed by examining the IgG1/IgG2α ratio at two and three weeks post-immunization. S.c. immunization resulted in a reduction in the IgG1/IgG2α ratio (Th1 tendency), whereas i.n. immunization caused a shift from Th1 to Th2. Moreover, immunohistochemistry showed that Em-TSP1 and -TSP3 were extensively located on the surface of E. multilocularis cysts, protoscoleces and adult worms with additional expression of Em-TSP3 in the inner part of protoscoleces and oncospheres. CONCLUSIONS: Our study indicated that i.n. administration of rEm-TSP3 with CpG is able to induce both systemic and local immune responses and thus provides significant protection against AE.

Complete coding sequence, sequence analysis and transmembrane topology modelling of Trypanosoma brucei rhodesiense putative oligosaccharyl transferase (TbOST II).

The partial nucleotide sequence of putative Trypanosoma brucei rhodesiense oligosaccharyl transferase gene was previously reported. Here, we describe the determination of its full-length nucleotide sequence by Inverse PCR (IPCR), subsequent biological sequence analysis and transmembrane topology modelling. The full-length DNA sequence has an Open Reading Frame (ORF) of 2406 bp and encodes a polypeptide of 801 amino acid residues. Protein and DNA sequence analyses revealed that homologues within the genome of other kinetoplastid and various origins exist. Protein topology analysis predicted that Trypanosoma brucei rhodesiense putative oligosaccharyl transferase clone II (TbOST II) is a transmembrane protein with transmembrane helices in probably an N(cytosol)-C(cytosol) orientation. Data from the GenBank database assembly and sequence analyses in general clearly state that TbOST II is the STT3 subunit of OST in T.b. rhodesiense that necessitates further characterisation and functional studies with RNAi. TbOST II sequence had been deposited in the GenBank (accession number GU245937).

Identification of genetic loci affecting the establishment and development of Echinococcus multilocularis larvae in mice.

Alveolar echinococcosis (AE) is a severe hepatic disorder caused by larval infection by the fox tapeworm Echinococcus multilocularis. The course of parasitic development and host reactions are known to vary significantly among host species, and even among different inbred strains of mice. As reported previously, after oral administration of parasite eggs, DBA/2 (D2) mice showed a higher rate of cyst establishment and more advanced protoscolex development in the liver than C57BL/6 (B6) mice. These findings strongly suggest that the outcome of AE is affected by host genetic factor(s). In the present study, the genetic basis of such strain-specific differences in susceptibility/resistance to AE in murine models was studied by whole-genome scanning for quantitative trait loci (QTLs) using a backcross of (B6×D2)F(1) and D2 mice with varying susceptibility to E. multilocularis infection. For cyst establishment, genome linkage analysis identified one suggestive and one significant QTL on chromosomes (Chrs.) 9 and 6, respectively, whereas for protoscolex development, two suggestive and one highly significant QTLs were detected on Chrs. 6, 17 and 1, respectively. Our QTL analyses using murine AE models revealed that multiple genetic factors regulated host susceptibility/resistance to E. multilocularis infection. Moreover, our findings show that establishment of the parasite cysts in the liver is affected by QTLs that are distinct from those associated with the subsequent protoscolex development of the parasite, indicating that different host factors are involved in the host-parasite interplay at each developmental stage of the larval parasite. Further identification of responsible genes located on the identified QTLs could lead to the development of effective disease prevention and control strategies, including an intensive screening and clinical follow-up of genetically high-risk groups for AE infection.

A schizont-derived protein, TpSCOP, is involved in the activation of NF-kappaB in Theileria parva-infected lymphocytes.

Theileria parva is a tick-transmitted intracellular protozoan parasite that causes East Coast fever, a fatal bovine lymphoproliferative disease. The molecular mechanisms that underlie host cell transformation by T. parva schizonts have been studied extensively, and it is known that the nuclear factor-kappa B (NF-kappaB) is activated in schizont-infected cells, making T. parva-transformed cells resistant to apoptosis. However, the mechanism by which the parasite triggers the activation of NF-kappaB remains enigmatic. In the present study, we biochemically characterized a novel protein, which we termed TpSCOP (T. parvaschizont-derived cytoskeleton-binding protein), which is expressed in the schizont stage of T. parva. TpSCOP was shown to interact with F-actin in vitro. Expression of TpSCOP in a murine lymphocytic cell line resulted in the activation of NF-kappaB signaling pathways, leading to apoptosis resistance. The activation of mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), was also detected. Furthermore, the introduction of TpSCOP into T. parva-infected cells also enhanced the activation of NF-kappaB. This is the first report to demonstrate that a parasite-derived molecule has the ability to activate the host NF-kappaB pathway. Based on these results, TpSCOP likely plays an important role in apoptosis inhibition during Theileria infection.

Overproduction of the pro-apoptotic molecule, programmed cell death 5, in Toxoplasma gondii leads to increased apoptosis of host macrophages.

We established a recombinant strain of Toxoplasma gondii that overexpressed programmed cell death 5 (TgPDCD5), in order to evaluate the role of endogenous TgPDCD5 in macrophage apoptosis during T. gondii infection. Immunofluorescence microscopy revealed that overproduced TgPDCD5 with a hemagglutinin tag was localized in the cytosol, which was consistent with the localization of endogenous TgPDCD5. The induced TgPDCD5-HA was recognized as an additional band by Western blot analysis, indicating successful overexpression of TgPDCD5. Secretion and release of TgPDCD5 by the parasite was also up-regulated in a time-dependent manner, which reflected its overproduction. Apoptosis due to parasite infection and interferon-gamma treatment was significantly up-regulated by the overexpression of TgPDCD5. These results suggest that endogenous TgPDCD5 plays a role in macrophage apoptosis during T. gondii infection.

Evaluation of Echinococcus multilocularis tetraspanins as vaccine candidates against primary alveolar echinococcosis.

Echinococcus multilocularis causes an important zoonotic cestode disease. The metacestode stage proliferates in the liver of intermediate hosts including human and rodents and forms multiple cysts. Recently, members of a transmembrane protein tetraspanin (TSP) family have been used as vaccines against schistosomosis, or as diagnostic antigens for cysticercosis. In this study, seven tetraspanins of E. multilocularis, designated as TSP1 to TSP7, were evaluated for their protective potential against primary alveolar echinococcosis. The large extracellular loop (LEL) region of these tetraspanins was cloned from a full-length enriched cDNA library of E. multilocularis metacestodes and expressed in Escherichia coli as a fusion protein with thioredoxin. Recombinant TSPs were applied as vaccines against an E. multilocularis primary experimental infection in BALB/c mice. Cyst lesions in the livers of vaccinated and non-vaccinated mice were counted. The cyst lesion reduction rates induced by the seven tetraspanins in vaccinated vis-à-vis non-vaccinated mice were: 87.9%, 65.8%, 85.1%, 66.9%, 73.7%, 72.9% and 37.6%. Vaccination conferred protective rates to mice ranging from 0% (TSP5, 6, 7) to maximally 33% (TSP1, 3). The results indicated that recombinant tetraspanins have varying protective effects against primary alveolar echinococcosis and could be used in vaccine development.

Molecular cloning and characterization of a T24-like protein in Echinococcus multilocularis.

One tetraspanin, designated as E24, was cloned from a full-length enriched vector-capping cDNA library of Echinococcus multilocularis metacestode. The amino acid sequence and phylogenetic analysis suggested that E24 is a T24-like protein. The crucial, functional large extracellular loop (LEL) domain of E24 was expressed and characterized using a polyclonal antiserum by Western blot and immunohistochemistry. The results showed that anti-recombinant-E24 (anti-recE24) antibody can specifically recognize approximately 25 kDa recombinant protein and 25 kDa cyst-extracted antigen; the germinal layer of both the protoscolex-free and protoscolex-formed cysts were intensely labeled by immunofluorescent antibody. This study revealed that E24 is an antigenic, germinal layer-located protein of E. multilocularis metacestode, implying for its potential in diagnostic and vaccine development.

Identification and molecular characterization of a novel stage-specific surface protein of Trypanosoma congolense epimastigotes.

The cattle pathogen Trypanosoma congolense expresses life cycle stage-specific surface molecules involved in adaptation to different host and vector environments. Here we report the discovery and molecular characterization of a novel stage-specific GPI-anchored surface glycoprotein that is selectively expressed in the epimastigote (EMF) life cycle stage of T. congolense. Culture supernatants of EMF but not of procyclic culture forms (PCFs) promoted adhesion of PCF parasites in an in vitro assay. Biosynthetic labeling experiments showed that these EMF culture supernatants contained a 100kDa trypanosome-derived protein that was not present in supernatants from PCF. We named this molecule "congolense epimastigote-specific protein" (CESP). The gene encoding CESP was isolated from an EMF cDNA library after immunoscreening. The multicopy gene had a 2070-bp open reading frame that encodes a polypeptide of 689 amino acids with a predicted mass of 72.9kDa. The discrepancy between the predicted (72.9kDa) and observed (100kDa) masses may be explained partially by glycosylation of the molecule which has six potential N-glycosylation sites and a predicted GPI anchor. Indeed, metabolic labeling of CESP with [(3)H] ethanolamine revealed that CESP was a GPI-anchored protein. Confocal laser scanning microscopy showed that CESP was expressed only on the surface of the EMF stage of the parasite. The identification of CESP as a unique component of culture supernatants from EMF and that such supernatants can confer plastic-adhesive ability on PCF suggest that CESP is worth further investigation as an adhesion molecule that perhaps allows T. congolense EMF to adhere to the tsetse proboscis.

Programmed Cell Death 5 from Toxoplasma gondii: a secreted molecule that exerts a pro-apoptotic effect on host cells.

Although parasite-infected host cells become resistant to apoptosis, uninfected bystander cells undergo apoptosis during Toxoplasma gondii infection. The Programmed Cell Death 5 (TgPDCD5) gene, a homologue of the human apoptosis-related molecule, was cloned from a T. gondii full-length cDNA database and subsequently characterized. The native TgPDCD5 was located in the cytosol and also detected in the secreted fraction. Immuno-electron microscopic analysis showed TgPDCD5 was primarily located close to the rhoptries or vesicle-like structures near the surface membrane of the parasite. Studies using recombinant TgPDCD5 (rTgPDCD5) demonstrated that host cells internalize the molecule in a heparan sulfate proteoglycan-binding motif-dependent manner. Furthermore, the addition of rTgPDCD5 to culture medium resulted in the enhancement of host-cell apoptosis triggered by etoposide in macrophage cell line J774A.1 and leukemic cell line HL-60 cells. Additionally, rTgPDCD5 induced apoptosis in J774A.1 cells in the presence of IFN-gamma. This report is the first to identify a parasitic molecule of T. gondii that has a pro-apoptotic effect on host cells.

The presence of a short form of p53 in chicken lymphoblastoid cell lines during apoptosis.

To examine the roles of a short form of p53 in the regulation of apoptosis in chicken lymphoblastoid tumor cell lines derived from Marek's disease (MD) and avian leukosis (AL), the expressions of the p53 proteins were analyzed in these cell lines in which apoptosis was chemically induced. In MSB1-O derived from MD, the expression of a 40 kDa protein of p53 was decreased and that of a 32 kDa protein, a short form of p53, was increased during apoptosis induced by actinomycin D. In 1104B1 derived from AL, the expressions of 42 and 32 kDa of p53 were increased during the apoptosis. The short form of p53 was undetectable in these cell lines when apoptosis was blocked by the pretreatment with endonuclease inhibitor, Zn2+, protease inhibitors, TPCK and TLCK, or caspase inhibitor, Z-VAD-FMK. In the transcriptional level, the expressions of bcl-2 and IAP were decreased in these cell lines during actinomycin D-induced apoptosis, but no change was detected in the expression level of p53. These results suggest that, in these chicken tumors, the short form of p53 could play a role in the initiation of apoptosis induced by the chemotherapeutic compound, and that the regulation of its expression may be important for the maintenance of transformation status.

The presence of the p53 transcripts with truncated open reading frames in Marek's disease tumor-derived cell lines.

Several kinds of the p53 transcripts in which their open reading frames (ORFs) were truncated (ranging from 101 to 765 bp) were identified in Marek's disease (MD)-derived tumor cell lines as well as avian leukosis- and reticuloendotheliosis-derived ones, detected by nested RT-PCR and subsequent nucleotide sequence analysis. In these ORFs, regions encoding the proline-rich and DNA-binding domains of the p53 protein were frequently deleted, and many of these deletions were found to cause frame shift. Western blot analysis using anti-p53 monoclonal antibodies revealed that multiple p53 isoform proteins with various molecular weights including 45-46, 35 and 28 kDa were expressed in these tumor cell lines, though the p53 protein with a molecular weight of 49 kDa was detected in chicken embryo fibroblasts transformed by the SV40 T antigen as a control. Since no deletions were found in the p53 gene of these MD tumor cell lines, truncations in the p53 ORFs observed in this study might result from alternative splicing of the p53 gene.