Role of Mtd/Bok in normal and neoplastic B-cell development in the bursa of Fabricius.

B-cell development in the bursa of Fabricius is accompanied by extensive apoptotic cell death. Apoptosis, however, is suppressed during c-myc-induced neoplasia. The experiments described here suggest that Mtd/Bok may drive apoptosis during normal development, and that this activity is blocked during myc-induced tumorigenesis. Bursal Mtd/Bok expression increases during development, correlating with the onset of intense, spontaneous apoptosis after hatching. Two isoforms of Mtd/Bok were characterized: WT-chMtd/Bok, found predominantly in the mitochondria and a less abundant form, lacking the presumptive transmembrane domain, Mtd/Bok deltaTM, found predominantly in the cytosol. Over-expression of Mtd/Bok deltaTM in a bursal lymphoma-derived cell line, DT40, reduced mitochondrial function and sensitized DT40 cells to apoptotic stimuli, while WT-chMtd/Bok had a diminished phenotype in these cells. In contrast, retroviral transduction of bursal stem cells with WT-chMtd/Bok ablated normal stem cell function in transplantation experiments, and produced extensive apoptosis in myc-induced pre-neoplastic bursal populations, but not in tumor cells.

Reduced Myc overexpression and normal B-cell differentiation mediate resistance to avian leukosis virus lymphomagenesis.

Avian leukosis virus (ALV) induces bursal lymphoma in tumor-susceptible chicken strains after proviral integration within the c-myc gene, and subsequent expansion of Myc-overexpressing lymphocytes within transformed follicles. Line 6(3) strain chickens are resistant to ALV tumorigenesis, largely failing to develop Myc-transformed follicles, although they show similar levels of ALV infection and integration as lymphoma-susceptible strains. Immunohistochemical analysis determined that the transformed follicles that do arise in lymphoma-resistant birds show much lower and more variable Myc overexpression than those of susceptible birds. This reduced Myc overexpression fails to block B-cell differentiation in resistant birds, while high Myc consistently blocks development at a late embryo stage in susceptible birds. This failure of Myc to block differentiation results in a normal pattern of posthatching bursal emigration in resistant transformed follicles, while transformed follicles of susceptible birds grow rapidly due to blocked emigration. Forced Myc overexpression produces transformed follicles in resistant birds, indicating that resistant lymphocytes can tolerate high Myc expression. The coding sequence and expression of the endogenous c-myc gene is the same in resistant and susceptible birds, suggesting that genetic resistance is instead mediated by reduced ALV LTR enhancer-driven transcription in the target lymphocytes of resistant birds.

Silent point mutation in an avian retrovirus RNA processing element promotes c-myb-associated short-latency lymphomas.

The avian leukosis virus DeltaLR-9 causes a high frequency of B-cell lymphomas within weeks after injection into 10-day-old chicken embryos. These lymphomas result from proviral integrations into the oncogene c-myb. In contrast, LR-9, which lacks the 42-nucleotide gag gene deletion of DeltaLR-9, does not cause a high frequency of c-myb-associated short-latency lymphomas. Although viral replication rates and spliced env mRNA levels were found to be similar for both viruses, DeltaLR-9 exhibited an increase in readthrough transcription compared to LR-9. The DeltaLR-9 deletion is located in the region of the gag gene corresponding to the matrix (MA) protein as well as in the negative regulator of splicing (NRS) element. To test whether disruption of the NRS or of the MA protein was responsible for inducing short-latency lymphomas, we generated viruses with NRS point mutations that maintained the wild-type Gag amino acid sequence. One of the mutant viruses induced an even higher incidence than DeltaLR-9 of short-latency lymphomas with viral integrations into c-myb. Thus, we propose that disruption of the NRS sequence promotes readthrough transcription and splicing to the downstream myb gene, causing overexpression of a slightly truncated Myb protein, which induces short-latency tumors.

Functional genomic analysis reveals distinct neoplastic phenotypes associated with c-myb mutation in the bursa of Fabricius.

Avian retroviral integration into the c-myb locus is casually associated with the development of lymphomas in the bursa of Farbricius of chickens; these arise with a shorter latency than bursal lymphomas caused by deregulation of c-myc. This study indicates that c-myb mutation in embryonic bursal precursors leads to an oligoclonal population of developing bursal follicles, showing a variable propensity to form a novel lesion, the neoplastic follicle (NF). About half of such bursas rapidly developed lymphomas. Detection of changes in gene expression, during the development of neoplasms, was carried out by cDNA microarray analysis. The transcriptional signature of lymphomas with mutant c-myb was more limited than, and only partially shared with, those of bursal lymphomas caused by Myc or Rel oncogenes. The c-myb-associated lymphomas frequently showed overexpression of c-myc and altered expression of other genes involved in cell cycle control and proliferation-related signal transduction. Oligoclonal, NF-containing bursas lacked detectable c-myc overexpression and demonstrated a pattern of gene expression distinct from that of normal bursa and partially shared with the short-latency lymphomas. This functional genomic analysis uncovered several different pathways of lymphomagenesis by oncogenic transcription factors acting in a B-cell lineage.