A novel, somatic, transforming mutation in the extracellular domain of Epidermal Growth Factor Receptor identified in myeloproliferative neoplasm.

We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2V617F Polycythaemia Vera (PV). This substitution affects a conserved cysteine residue in EGFR domain 2 and leads to the formation of a ligand-independent covalent receptor dimer, associated with increased transforming potential. Aberrant signalling from the EGFRC329R receptor is cell type-dependent and in the TF1.8 erythroid cell line expression of this mutant suppresses EPO-induced differentiation. Clonal analysis shows that the dominant JAK2V617F-positive clone in this PV patient harbors EGFRC329R, thus this mutation may contribute to clonal expansion. Somatic mutations affecting other ERBB and related receptor tyrosine kinases are observed in myeloproliferative neoplasms (MPN), and we show elevated EGFR levels in MPN samples, consistent with previous reports. Thus activation of this group of receptors, via multiple mechanisms, may contribute to clonal growth and survival of the JAK2V617F disease clone in MPN.

Different temporal expression of immunodominant Ro60/60 kDa-SSA and La/SSB apotopes.

Opsonization of apoptotic cardiocytes by maternal anti-Ro/SSA and anti-La/SSB antibodies contributes to tissue injury in the neonatal lupus syndrome. The objective of the current study was to quantify the surface membrane expression of Ro/La components during different phases of apoptosis and map the Ro/La apotopes (epitopes expressed on apoptotic cells) bound by cognate antibodies. Multi-parameter flow cytometry was used to define early and late apoptotic populations and their respective binding by monospecific anti-Ro and anti-La IgGs. Anti-Ro60 bound specifically to early apoptotic Jurkat cells and remained accessible on the cell surface throughout early and late apoptosis. In contrast, anti-La bound exclusively to late apoptotic cells in experiments controlled for non-specific membrane leakage of IgG. Ro52 was not accessible for antibody binding on either apoptotic population. The immunodominant NH2-terminal and RNA recognition motif (RRM) epitopes of La were expressed as apotopes on late apoptotic cells, confirming recent in vivo findings. An immunodominant internal epitope of Ro60 that contains the RRM, and is recognized by a majority of sera from mothers of children with congenital heart block (CHB) and patients with primary Sjögren's syndrome, was also accessible as an apotope on early apoptotic cells. The distinct temporal expression of the immunodominant Ro60 and La apotopes indicates that these intracellular autoantigens translocate independently to the cell surface, and supports a model in which maternal antibody populations against both Ro60 and La apotopes act in an additive fashion to increase the risk of tissue damage in CHB.

Apolipoprotein-D: a novel cellular marker for HGPIN and prostate cancer.

BACKGROUND: High grade prostatic intraepithelial neoplasia (HGPIN) is a putative pre-malignant lesion of the prostate. While apolipoprotein-D (Apo-D), an androgen-regulated hydrophobic transporter protein, is expressed in prostate tumors, its expression in HGPIN is unknown. METHODS: Immunoreactivity for Apo-D and another androgen-regulated protein, prostate specific antigen (PSA), was investigated in 64 radical prostatectomy tissues by video image analysis. RESULTS: Eighty two percent of prostatectomy specimens demonstrated moderate to strong Apo-D immunoreactivity in areas of HGPIN. In comparison, weak Apo-D immunoreactivity was observed in non-malignant areas in only 24% of specimens. The median (range) percentage cellular area of HGPIN immunopositive for Apo-D (9.7%, 0-42.9), and the cellular concentration of Apo-D (MIOD 3.1, 0-13.3), were intermediate between that of normal (area 0%, 0-53.5%, MIOD 0, 0-12.6) and early stage prostate cancer tissues (area 29.2%, 0-90.8%, MIOD 6.7, 0-28.1). This increase in Apo-D expression from non-malignant, through HGPIN to prostate cancer was statistically significant (P < 0.001), and contrasted with the decrease observed in PSA staining between adjacent areas of normal glands, HGPIN, and cancer (P = 0.026). CONCLUSIONS: The presence of high levels of immunoreactive Apo-D in HGPIN and prostate cancer, but not in non-malignant epithelial cells, is consistent with HGPIN being an intermediate lesion in the transition to prostate cancer, and suggests that cellular Apo-D expression is a marker of malignant transformation of the prostate.

Establishing lysogenic transcription in the temperate coliphage 186.

A single-copy chromosomal reporter system was used to measure the intrinsic strengths and interactions between the three promoters involved in the establishment of lysogeny by coliphage 186. The maintenance lysogenic promoter p(L) for the immunity repressor gene cI is intrinsically approximately 20-fold weaker than the lytic promoter p(R). These promoters are arranged face-to-face, and transcription from p(L) is further weakened some 14-fold by the activity of p(R). Efficient establishment of lysogeny requires the p(E) promoter, which lies upstream of p(L) and is activated by the phage CII protein to a level comparable to that of p(R). Transcription of p(E) is less sensitive to converging p(R) transcription and raises cI transcription at least 55-fold. The p(E) promoter does not occlude p(L) but inhibits lytic transcription by 50%. This interference is not due to bound CII preventing elongation of the lytic transcript. The p(E) RNA is antisense to the anti-immune repressor gene apl, but any role of this in the establishment of lysogeny appears to be minimal.

The CII protein of bacteriophage 186 establishes lysogeny by activating a promoter upstream of the lysogenic promoter.

We have shown previously that the cII gene product of the non-lambdoid temperate bacteriophage 186 is required for the establishment of lysogeny. We show here that CII, a potential helix-turn-helix DNA-binding protein, establishes lysogeny by activating a promoter (PE) which spans the apl/cII intergenic region, upstream of the lysogenic promoter, PL. The start site of the PE transcript (+1) has been mapped by primer extension and we have identified the CII binding determinants at PE by DNase I footprinting. CII binds to inverted repeat sequences separated by two turns of the helix, with binding half-sites centred at the 38 and -58 positions of PE. Oligomerisation studies with purified CII protein indicate that a CII tetramer may be the species that binds to this site. We also show that PE is subject to direct negative feedback by the CI repressor.