Induction of pRb degradation by the human papillomavirus type 16 E7 protein is essential to efficiently overcome p16INK4a-imposed G1 cell cycle Arrest.

It has previously been shown that the E7 protein from the cutaneous human papillomavirus type 1 (HPV1), which is associated with benign skin lesions, binds the product of the tumor suppressor gene retinoblastoma (pRb) with an efficiency similar to that of the E7 protein from the oncogenic HPV type 16. Despite this ability, HPV1 E7 does not display any activity in transforming primary cells. In addition, the two viral proteins differ in their mechanisms of targeting pRb. HPV16 E7 promotes pRb destabilization, while cells expressing HPV1 E7 do not show any decrease in pRb levels. In this study, we show that HPV1 E7, in contrast to HPV16 E7, has only a weak activity to neutralize the effect of cyclin-dependent kinase inhibitor p16INK4a. By generation of HPV1/16 E7 chimeric proteins, we have identified a central motif in the two E7 proteins, which determines their different abilities to overcome the p16INK4a-mediated cell cycle arrest. This motif is located downstream of the pRb-binding domain and comprises only three amino acids in HPV16 E7. Swapping this central motif in the two viral proteins causes an exchange of their activities involved in circumventing the inhibitory function of p16INK4a. Most importantly, our data show that the efficiency of the E7 proteins in neutralizing the inhibitory effect of p16INK4a correlates with their ability to promote pRb degradation.

Induction of S phase and apoptosis by the human papillomavirus type 16 E7 protein are separable events in immortalized rodent fibroblasts.

The HPV16 E7 oncoprotein neutralizes several cell cycle checkpoints, favouring the entry of quiescent cells into S phase. This activity is mediated in part by association of E7 with the pocket proteins and consequent activation of E2F transcription factors. In addition, HPV16 E7 protein is able to promote apoptosis. In this study we demonstrate that the ability to induce apoptosis is a common property of E7s belonging to both benign and malignant HPV types. The E7-induced apoptosis is mediated by inactivation of pRb, whilst neutralization of the other two pRB-related proteins, p107 and 130, is not sufficient to trigger apoptosis. Moreover, we show that certain point mutations in the conserved region 1 (CR1) of HPV16 E7 abolish the induction of apoptosis without altering the ability to stimulate S phase. Thus, these two E7-mediated cellular events, apoptosis and S phase entry, can be separated in immortalized rodent fibroblasts. Our findings demonstrate that the E7-mediated pRb destabilization is not required for its ability to drive quiescent cells into S phase and to induce apoptosis. Finally, expression of E7 proteins in NIH3T3, which lack a functional p19ARF, does not lead to p53 accumulation, indicating that the E7 impacts upon additional cellular pathways to promote apoptosis.

Wnt signaling stabilizes the dual-function protein beta-catenin in diverse cell types.

The Wnt proteins constitute a large family of secreted signaling factors that performed a wide variety of inductive and regulatory functions in embryonic and postnatal development. In mammals, these include crucial roles in morphogenesis of the central nervous system, kidneys, limbs, and mammary glands. In recent years, much progress has been made in identifying components of the intracellular Wnt-1 signal transduction pathway through studies in Drosophila, C. elegans, Xenopus, and mammalian systems. Several features of this pathway are remarkably well conserved in evolution. A key component of the Wnt pathway is the cytoplasmic protein beta-catenin, whose stability is increased as a result of Wnt signaling. Although morphological effects of Wnt-1 in cell culture are seen in only a limited number of cell lines, we show here that responsiveness to Wnt-1 at the biochemical level is a common property of both epithelial and mesenchymal cells. The increased abundance of beta-catenin may have at least two functional consequences, depending on the subcellular localization of the protein. In some cell lines that respond to Wnt-1, there is a significant increase in the beta-catenin fraction associated with the plasma membrane, where the protein acts as a component of cell-cell adhesive junctions. In other cell types, the major effect of Wnt signaling is an increase in the cytosolic pool of beta-catenin. Increased abundance of this pool has recently been correlated with entry of beta-catenin into the nucleus, where the protein forms complexes with Tcf transcription factors and is thought to modulate the expression of specific genes. The dual consequences of Wnt signaling on cell adhesion and/or gene expression provide at least two potential mechanisms by which this key pathway can function in the regulation of morphogenesis.

Transformation by Wnt family proteins correlates with regulation of beta-catenin.

Several members of the Wnt family of secreted factors are strongly implicated as regulators of mammary cell growth and differentiation. To investigate Wnt signaling in mammary cells, we have assessed the abilities of 10 different Wnt genes to cause transformation of C57MG mammary epithelial cells and in parallel studied their effects on beta-catenin, a component of the Wnt-1 signaling pathway. Autocrine transforming potential was tested by expression of Wnt proteins in C57MG cells, and paracrine effects were evaluated by coculture of C57MG cells with fibroblasts secreting different Wnt proteins. Western blotting confirmed the expression of each Wnt protein in the relevant cell lines. Activities of the 10 Wnts tested were divisible into three groups. Wnt-1, Wnt-2, Wnt-3, and Wnt3a induced strong transformation and an elongated refractile cell morphology. Wnt-6 and Wnt-7a produced weak morphological changes. Wnt-4, Wnt-5a, Wnt-5b, and Wnt-7b had no effect at all on C57MG morphology. Analysis of beta-catenin levels showed that the transforming Wnts induced accumulation of cytosolic beta-catenin, whereas nontransforming Wnts did not. These result demonstrate that several Wnt family members are capable of elevating beta-catenin levels and suggest that their signaling pathways share intracellular signaling components. The correlation between increased cytosolic beta-catenin levels and C57MG transformation supports a role for beta-catenin in transformation of these cells. These data also imply the existence of receptors that respond to certain Wnt proteins but not to others.

Velocity vector reconstruction for color flow Doppler: experimental evaluation of a new geometrical method.

Ultrasonic color flow mapping (CFM) is employed for the diagnosis of a variety of cardiovascular abnormalities. But CFM permits only the analysis of the in-beam component of the velocity distribution. A reconstruction method based on a geometrical approach was developed to calculate the two-dimensional, in-plane velocity vector using the information of two independent CFM measurements obtained from two closely spaced transducers. However, with the CFM measurements being limited by the noise and quantified in a limited number of velocity values, the accuracy of the velocity vector reconstruction is not satisfactory. A simple filtering process was applied to improve the results. The method was tested with simulations and a series of in vitro measurements. The results of the simulations were compared with analytical solutions. The in vitro measurements were compared with the results of laser Doppler anemometry (LDA) and theoretical calculations. The results of in vivo measurements were compared with theoretical calculations based on Womersley's theory. The comparison showed good applicability of the method to different flow fields. The flow vector reconstructed without filtering had an error of more than 20%; whereas our method, including the filtering process, had an error of less than 10%, in comparison with the theoretical or the LDA results. The method could be extended to three-dimensional flows.

mRNAs for activin receptors II and IIB are expressed in mouse oocytes and in the epiblast of pregastrula and gastrula stage mouse embryos.

Activin is a potent inducer of mesoderm in frog embryos. We showed previously that in the mouse, activin beta A is expressed in the uterine decidua near the embryo before and during the first appearance of mesoderm (E4.5-E6.5). Here, using Northern blotting and in situ hybridization, we show that mouse oocytes, E6.5 and E7.5 embryos, and E6.5 and E7.5 decidua contain mRNAs for both activin receptors type II and IIB. The expression of activin receptor type IIB is particularly strong in embryonic ectoderm apparent at E5.5 and continuing through E8.5. These results support the hypothesis that activin derived from the decidua promotes development of mesoderm in the period E5.5-E6.5.

Dependence of position-effect variegation in Drosophila on dose of a gene encoding an unusual zinc-finger protein.

Position-effect variegation is the inactivation in some cells of a gene translocated next to heterochromatin, the region of the chromosome that is permanently condensed. The number of copies of the Drosophila gene Suvar(3)7 is a dose-limiting factor in this phenomenon, and seems from its sequence that it encodes a protein with five widely spaced zinc-fingers. This novel arrangement of zinc-fingers could help in packaging the chromatin fibre into heterochromatin, and also reflect a novel method of controlling the expression from DNA domains.