Neurotrophin responsiveness is differentially regulated in neurons and precursors isolated from the developing striatum.

Sequential exposure to members of the neurotrophin family, nerve growth factor (NGF), neurotrophin-type 3 (NT3), brain-derived neurotrophic factor (BDNF), and neurotrophin-type 4 (NT4), determines the generation, survival, and maturation of developing neurons. The effects of neurotrophins depend on the stage of development and the target cell population. However, the nature of the responding cells is often unclear. In this study neurotrophin responsiveness was analyzed in murine embryonic striatal precursors and neurons. Individual neurotrophin-responsive cells were identified based on activation of intracellular signaling pathways to the transcription factor CREB and were further characterized using differentiation-stage specific markers. A dramatic developmentally regulated decrease in BDNF responsiveness was observed: BDNF targeted more than 40% of striatal neurons at E14 but only 12% at E18. The percentage of NT3-responsive neurons also moderately decreased during development while no change was observed in the fraction of neuronal cells targeted by NT4 and NGF. A different type of developmental change was found in striatal precursors. BDNF, NT3, and NT4 each targeted about 15% of striatal precursors at E14 but no NGF responsive-precursors were detected at this age. In contrast, only NT3 and NGF could induce a response in precursor cells at E18. NGF-responsive precursors shared a distinct morphology with a large cell body and high levels of nestin expression. These results indicate that during striatal development, the regulation of neurotrophin responsiveness is different in neurons and precursor cells.

Identification of two distinct types of multipotent neural precursors that appear sequentially during CNS development.

Epidermal growth factor (EGF) and fibroblast growth factor (FGF)-2 control neural stem cell proliferation in vitro and the formation of neurospheres. Neurospheres contain precursors that respond to both EGF and FGF-2 (E/F cells). E/F cells appear to originate from cells that initially respond to FGF-2 only but undergo a transition in growth factor responsiveness during in vitro culturing. It is unclear whether a similar change in growth factor responsiveness of multipotent precursors takes place in vivo and how this may affect neural precursor properties. Here I provide evidence that FGF-2-responsive precursors and E/F cells appear sequentially during CNS development. This transition from the early precursors (FGF-2-responsive cells) to the late precursors (E/F cells) takes place between E14 and E18. The two types of precursors are morphologically and antigenically distinct. E/F cells are very large and show strong nestin immunoreactivity. Thus the putative neurosphere-forming E/F cells are present in vivo and their generation is developmentally programmed. Their unique morphology may provide a basis for their isolation.

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.

Calcium signalling--an overview.

Calcium (Ca2+) is an almost universal intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation. The ability of a simple ion such as Ca2+ to play a pivotal role in cell biology results from the facility that cells have to shape Ca2+ signals in the dimensions of space, time and amplitude. To generate the variety of observed Ca2+ signals, different cell types employ components selected from a Ca2+ signalling 'toolkit', which comprizes an array of signalling, homeostatic and sensory mechanisms. By mixing and matching components from the toolkit, cells can obtain Ca2+ signals that suit their physiology.

Fibroblast growth factor 2 (FGF-2) promotes acquisition of epidermal growth factor (EGF) responsiveness in mouse striatal precursor cells: identification of neural precursors responding to both EGF and FGF-2.

Epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) induce the proliferation of neural precursor cells isolated from specific regions of the embryonic and adult brain. However, the lineage relationship between the EGF- and FGF-2-responsive cells is unknown. In this study we used phosphorylation of the transcription factor cAMP response element-binding protein as a functional readout to identify cells responding to EGF and FGF-2. In primary cultures of mouse embryonic day 14 (E14) striatum, maintained in vitro for 24 hr, 12% of the cells responded to FGF-2, whereas no response to EGF could be detected. Seventy-five percent of these FGF-2-responsive cells were beta tubulin III (TuJ1)-positive neurons, and 25% expressed nestin, a marker for neuroepithelial precursors. After growth factor treatment for 6 d, a population of nestin-positive cells responding to both EGF and FGF-2 were identified. The 6-d-old cultures also contained a small number of TuJ1-positive cells that responded to FGF-2 only. Priming of striatal cells for 24 hr with FGF-2 but not with EGF was sufficient to induce the appearance of EGF- and FGF-2 responsive cells after only 2 d in vitro. Thus, neural precursor cells from the mouse E14 striatum initially responding to FGF-2 only acquire EGF responsiveness later during in vitro development. At this stage EGF and FGF-2 act on the same cells. The acquisition of EGF responsiveness is promoted by FGF-2.

The CXXC Zn binding motifs of the human papillomavirus type 16 E7 oncoprotein are not required for its in vitro transforming activity in rodent cells.

The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule. Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins. CR3 also contains a low affinity pRb binding site and is involved in the disruption of the E2F/Rb1 complex. E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3. However, the Ad E1A transforming activity appears to be independent of the presence of such domains. In fact, this viral protein exists in vivo as two different forms of 289 and 243 amino acids. The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity. We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs. A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed. The E7/E1A243 while not able to homodimerize in the S. cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A. It associates with the 'pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo fibroblasts. These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein configuration.

Cell cycle-dependent disruption of E2F-p107 complexes by human papillomavirus type 16 E7.

The human papillomavirus type 16 (HPV-16) E7 and adenovirus (Ad) E1A oncoproteins share a common pathway of transformation. They disrupt the cell cycle G1 phase-specific protein complex containing the E2F transcription factor and the regulatory protein Rb1, the retinoblastoma tumour suppressor gene product. In the G1 and S phases of the cell cycle, E7 and E1A bind two other cellular complexes containing the Rb1-related protein p107 and E2F. Ad E1A disrupts both complexes and releases active E2F. In contrast, HPV-16 E7, although it efficiently binds both E2F-p107 complexes, causes dissociation of the G1 phase complex only. Using chimeric proteins of HPV-16 E7 and Ad E1A we were able to demonstrate that the ability of E1A to disrupt both G1 and S phase E2F-p107 complexes is not due to the higher concentration of Ad E1A in the cell, but is an intrinsic property of the Ad E1A transforming region. These data suggest that E1A and E7 may function in cellular transformation in similar, but not identical ways.

Functional studies of E7 proteins from different HPV types.

We have performed comparative studies on the E7 proteins from malignant and non-malignant Human Papillomavirus types HPV 1, 6, 11, 16, 18, 33). GST/E7 fusion proteins from all these HPV types associate with Rb1, p107 and the cyclin A/CDK2 complex. As has been shown for Rb1, the association with p107 and Cyclin A was weaker for the 'low risk' HPV6 and 11 E7 proteins as compared to 'high risk' HPV16, 18 and 33 E7 proteins. In contrast the E7 protein of the benign type HPV1 bound Rb1, p107 and cyclin A with the same affinity as the 'high risk' E7 proteins. The affinities of the E7/Rb1 interaction have been confirmed in vivo by the 'two hybrid' method in the yeast Saccharomyces cerevisiae. Although HPV1 E7 showed the same affinity in vitro and in vivo for Rb1 as the high risk HPV E7s, it did not have the ability to activate the E2F-1 transcription factor inhibited by Rb1, nor did it have any transforming activity when coexpressed with activated ras in primary rodent cells.

Susceptibility of multidrug-resistant human T-lymphoblastoid CEM cell line to cell-mediated cytolysis.

The using of multidrug-resistant (MDR) cell variants represents one of the major obstacles to an effective cancer therapy based on the administration of cytotoxic compounds. In the present article we describe experimental procedures able to eradicate, in vitro, by using specific immunological reagent, MDR tumor cells. In an allogeneic cell system, natural killer (NK) and lymphokine activated killer (LAK) cells result effective against MDR variants of the human T-lymphoblastoid CEM cell line. Surprisingly effector cells discriminate in their lytic capacity target cells possessing a MDR phenotype. A direct relationship between the degree of relative resistance shown by target cells and cytotoxic level exerted by peripheral lymphocytes stimulated and non by IL-2 was observed. The preincubation of MDR cell variants with a monoclonal antibody (MoAb57) specific for an extramembranal epitope of P-glycoprotein induced, in presence of effector cells, a strong antibody-dependent cell-mediated cytolysis (ADCC). This phenomenon was observed only in MDR variants over-expressing in concomitance with drug-resistance high level of P-glycoprotein. In identical experimental conditions, drug-sensitive parental cells does not show valuable ADCC.