A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation.

Bruton's tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5'-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach.

Exposure to 900 MHz radiofrequency radiation induces caspase 3 activation in proliferating human lymphocytes.

In this study, the induction of apoptosis after exposure to 900 MHz radiofrequency radiation (GSM signal) was investigated by assessing caspase 3 activation in exponentially growing Jurkat cells and in quiescent and proliferating human peripheral blood lymphocytes (PBLs). The exposure was carried out at an average specific absorption rate of 1.35 W/kg in a dual wire patch cell exposure system where the temperature of cell cultures was accurately controlled. After 1 h exposure to the radiofrequency field, a slight but statistically significant increase in caspase 3 activity, measured 6 h after exposure, was observed in Jurkat cells (32.4%) and in proliferating human PBLs (22%). In contrast, no effect was detected in quiescent human PBLs. In the same experimental conditions, apoptosis was also evaluated in Jurkat cells by Western blot analysis and in both cell types by flow cytometry. To evaluate late effects due to caspase 3 activity, flow cytometry was also employed to assess apoptosis and viability 24 h after radiofrequency-radiation exposure in both cell types. Neither the former nor the latter was affected. Since in recent years it has been reported that caspases are also involved in processes other than apoptosis, additional cell cycle studies were carried out on proliferating T cells exposed to radiofrequency radiation; however, we found no differences between sham-exposed and exposed cultures. Further studies are warranted to investigate the biological significance of our findings of a dose-response increase in caspase 3 activity after exposure to radiofrequency radiation.

E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis.

The retinoblastoma protein (pRB) and its two relatives, p107 and p130, regulate development and cell proliferation in part by inhibiting the activity of E2F-regulated promoters. We have used high-density oligonucleotide arrays to identify genes in which expression changed in response to activation of E2F1, E2F2, and E2F3. We show that the E2Fs control the expression of several genes that are involved in cell proliferation. We also show that the E2Fs regulate a number of genes involved in apoptosis, differentiation, and development. These results provide possible genetic explanations to the variety of phenotypes observed as a consequence of a deregulated pRB/E2F pathway.

BCR-ABL prevents c-jun-mediated and proteasome-dependent FUS (TLS) proteolysis through a protein kinase CbetaII-dependent pathway.

The DNA binding activity of FUS (also known as TLS), a nuclear pro-oncogene involved in multiple translocations, is regulated by BCR-ABL in a protein kinase CbetaII (PKCbetaII)-dependent manner. We show here that in normal myeloid progenitor cells FUS, although not visibly ubiquitinated, undergoes proteasome-dependent degradation, whereas in BCR-ABL-expressing cells, degradation is suppressed by PKCbetaII phosphorylation. Replacement of serine 256 with the phosphomimetic aspartic acid prevents proteasome-dependent proteolysis of FUS, while the serine-256-to-alanine FUS mutant is unstable and susceptible to degradation. Ectopic expression of the phosphomimetic S256D FUS mutant in granulocyte colony-stimulating factor-treated 32Dcl3 cells induces massive apoptosis and inhibits the differentiation of the cells escaping cell death, while the degradation-prone S256A mutant has no effect on either survival or differentiation. FUS proteolysis is induced by c-Jun, is suppressed by BCR-ABL or Jun kinase 1, and does not depend on c-Jun transactivation potential, ubiquitination, or its interaction with Jun kinase 1. In addition, c-Jun-induced FUS proteasome-dependent degradation is enhanced by heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and depends on the formation of a FUS-Jun-hnRNP A1-containing complex and on lack of PKCbetaII phosphorylation at serine 256 but not on FUS ubiquitination. Thus, novel mechanisms appear to be involved in the degradation of FUS in normal myeloid cells; moreover, the ability of the BCR-ABL oncoprotein to suppress FUS degradation by the induction of posttranslational modifications might contribute to the phenotype of BCR-ABL-expressing hematopoietic cells.

Multiple signaling pathways of the insulin-like growth factor 1 receptor in protection from apoptosis.

The type 1 insulin-like growth factor receptor (IGF-1R), activated by its ligands, protects several cell types from a variety of apoptotic injuries. The main signaling pathway for IGF-1R-mediated protection from apoptosis has been previously elucidated and rests on the activation of phosphatidylinositol 3-kinase, Akt/protein kinase B, and the phosphorylation and inactivation of BAD, a member of the Bcl-2 family of proteins. In 32D cells (a murine hemopoietic cell line devoid of insulin receptor substrate 1 [IRS-1]), the IGF-1R activates alternative pathways for protection from apoptosis induced by withdrawal of interleukin-3. One of these pathways leads to the activation of mitogen-activated protein kinase, while a third pathway results in the mitochondrial translocation of Raf and depends on the integrity of a group of serines in the C terminus of the receptor that are known to interact with 14.3.3 proteins. All three pathways, however, result in BAD phosphorylation. The presence of multiple antiapoptotic pathways may explain the remarkable efficacy of the IGF-1R in protecting cells from apoptosis.

Resistance to apoptosis in CTLL-2 cells overexpressing B-Myb is associated with B-Myb-dependent bcl-2 induction.

Transcriptional regulators of the Myb family play important roles in cell proliferation, differentiation, and survival. To investigate the role of Myb proteins in the regulation of apoptosis, we studied the apoptotic response of interleukin 2-dependent CTLL-2 cells stably transfected with B-Myb. B-Myb-overexpressing cells showed a diminished cytokine dependence and were resistant to apoptosis induced by doxorubicin, ceramide, and dexamethasone. Overexpression of B-Myb was associated with enhanced expression of bcl-2, which was dependent, at least in part, on increased transcription. In transient transfection assays in T-lymphoblastic cells, B-Myb was able to stimulate the promoter activity of the bcl-2 5' flanking region linked to the chloramphenicol acetyltransferase reporter gene. A segment of the bcl-2 promoter (nucleotides +34 to +58 relative to the transcription initiation site) contained a putative Myb-binding site and was shown to specifically interact with B-Myb and to confer B-Myb responsiveness to a bcl-2/chloramphenicol acetyltransferase reporter construct. These results indicate that B-Myb promotes T cells survival by enhancing the expression of bcl-2 and identify bcl-2 as a B-Myb target gene regulated in a DNA binding-dependent manner.

Inhibition of proteasome function prevents thymocyte apoptosis: involvement of ornithine decarboxylase.

We have previously shown that polyamine levels rapidly decrease in thymocytes undergoing apoptosis, and that ornithine decarboxylase increases early but too transiently to maintain elevated polyamine levels. These data led us to suppose that a precocious ornithine decarboxylase degradation might be responsible for the imbalance of polyamine metabolism. Ornithine decarboxylase is known to be degraded by the cytosolic 26S proteasome that plays an essential role in thymocyte apoptosis. In this paper we demonstrate that the inhibition of proteasome function preserves ornithine decarboxylase activity and prevents thymocytes from undergoing apoptosis after dexamethasone treatment. Since intracellular polyamine levels are also preserved, ornithine decarboxylase seems to be functionally active in maintaining polyamine homeostasis after proteasome inhibition in thymocytes. Our proposed role for the proteasome in quiescent cells upon an apoptotic stimulus is to degrade proteins like ornithine decarboxylase that are involved in the control of the cell cycle and cell survival.

TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis.

The leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effectors, some of which are essential for cell transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double-stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA-binding protein specifically induced by BCR/ABL. The peptide sequence of the affinity-purified protein was identical to that of the RNA-binding protein FUS (also called TLS). Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKCbetaII-dependent FUS phosphorylation. Moreover, suppression of PKCbetaII activity in BCR/ABL-expressing cells by treatment with the PKCbetaII inhibitor CGP53353, or by expression of a dominant-negative PKCbetaII, markedly impaired the ability of FUS to bind DNA. Suppression of FUS expression in myeloid precursor 32Dcl3 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte-colony stimulating factor receptor (G-CSFR) and downregulation of interleukin-3 receptor (IL-3R) beta-chain expression, and accelerated G-CSF-stimulated differentiation. Downregulation of FUS expression in BCR/ABL-expressing 32Dcl3 cells was associated with suppression of growth factor-independent colony formation, restoration of G-CSF-induced granulocytic differentiation and reduced tumorigenic potential in vivo. Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression.

Protection against apoptosis by monoamine oxidase A inhibitors.

Several lines of evidence have been accumulating indicating that an important role may be played by mitochondrial homeostasis in the initiation phase, the first stage of apoptosis. This work describes the results obtained by using different inhibitors of monoamine oxidases (MAO), i.e. pargyline, clorgyline and deprenyl, on mitochondrial integrity and apoptosis. Both pargyline and clorgyline are capable of protecting cells from apoptosis induced by serum starvation while deprenyl is ineffective. These data represent the first demonstration that MAO-A inhibitors may protect cells from apoptosis through a mechanism involving the maintenance of mitochondrial homeostasis.

Growth properties and growth factor responsiveness in skin fibroblasts from centenarians.

Human fibroblast cultures, which have a finite replicative lifespan in vitro, are the most widely used model for the study of senescence at the cellular level. An inverse relationship between replicative capability and donor age has been reported in human fibroblast strains. We studied the growth capacity of fibroblast primary cultures derived from people whose lifespan was as closer as possible to the expected maximum human lifespan, i.e. people over one hundred. Our data suggest that outgrowth of fibroblasts from biopsies, growth kinetics at different population doubling levels, capability to respond to a classical mitogenic stimulus (such as 20% serum) and a variety of growth factors, were remarkably similar in fibroblasts from centenarians and young controls. On the whole, our data challenge the tenet of a simple and strict relationship between in vivo aging and in vitro proliferative capability of human fibroblasts, at least at the individual level.

Autocrine nerve growth factor protects human keratinocytes from apoptosis through its high affinity receptor (TRK): a role for BCL-2.

Normal human keratinocytes synthesize and release nerve growth factor (NGF) and express both the low- and the high-affinity NGF receptor. Because NGF has been shown to rescue certain cell types from programmed cell death, we investigated the role of endogenous NGF in preventing keratinocyte apoptosis. We report here that apoptosis is induced in normal human keratinocytes in culture by blocking endogenous NGF signaling with either anti-NGF neutralizing antibody or K252, a specific inhibitor of the tyrosine kinase high-affinity NGF receptor. Apoptosis was assessed by DNA laddering, electron microscopy, and in situ nick end labeling technique. In anti-NGF-treated keratinocytes, the apoptotic process starts at 96 h, and is maximal at 120 h. After K252 treatment, apoptosis starts at 48 h and peaks at 120 h. Because the product of the bcl-2 proto-oncogene protects many cell types from apoptosis, we measured the levels of this protein in apoptotic keratinocytes. We found that both K252 and anti-NGF antibody strikingly downregulate bcl-2 expression, starting at 72 h. Furthermore, HaCat keratinocytes stably transfected with a plasmid containing bcl-2 cDNA fail to undergo apoptosis when treated with K252. These findings show that autocrine NGF acts as a survival factor for human keratinocytes in vitro through its high-affinity NGF receptor, possibly by maintaining constant levels of Bcl-2.

c-fos/c-jun expression and AP-1 activation in skin fibroblasts from centenarians.

In vitro replicative senescence is characterized by an irreversible growth arrest due to the inability of the cell to induce some key regulators of cell cycle progression, such as c-fos and AP-1, in response to mitogenic stimuli. In vitro replicative senescence and in vivo aging have been assumed to be two related phenomena, likely controlled by overlapping or interacting genes. As a corollary, fibroblasts from centenarians, which have undergone a long process of senescence in vivo should have very limited proliferative capability. On the contrary, in a previous work we found that fibroblasts from centenarians exhibited the same capacity to respond to different mitogenic stimuli as fibroblasts from young donors. Here we provide evidences that the well preserved proliferative response is likely due to the fact that some pivotal regulators- c-fos, c-jun and AP-1-are still fully inducible, despite a long process of in vivo senescence. Our data therefore suggest that in vivo and in vitro aging are separate phenomena whose possible relationships, if any, have to be ascertained very carefully.

Successful immunosenescence and the remodelling of immune responses with ageing.

In recent decades, major theoretical and technological advances have been achieved in the field of immunology. These have allowed the scientific community to analyse the immune system in a much more sophisticated manner than was possible even 20 years ago. Moreover, great theoretical changes have also occurred in gerontology-in particular, the hypothesis has been put forward that ageing and diseases are two different phenomena, and that successful ageing, i.e. ageing in good psychophysical conditions, is really possible for most humans and animals. Immunosenescence was then carefully investigated, either in selected healthy people of advanced age or in the oldest old people, such as healthy centenarians. The main results showed that most immune parameters are indeed well preserved even at this far advanced age. This paper deals with some of the most important theoretical problems of immunosenescence. An immunological tenet was that the most important phenomenon of immunosenescence is the involution of the thymus. In most textbooks and papers it is taken for granted that the thymus starts its involution immediately after puberty. When people aged 60-65 were considered old, it was not difficult to think that they could live for the rest of their life with a fully involuted thymus. The findings on centenarians challenge this tenet, as they have only a small reduction of T lymphocytes, and a relatively normal number of virgin and memory T cells, together with a functional T cell repertoire. Other observations reported here on centenarians, concerning the activity of B lymphocytes and the cytokine network, as well as those on the well-preserved innate immunity and the cells' capability of undergoing proliferation after appropriate stimuli, suggest that complex immune changes occur with age, but also indicate that we have to modify our attitude, to grasp the new scenario which is emerging. Immunosenescence can no longer be considered as a unidirectional deterioration, and this complex phenomenon is much better described by terms such as 'remodelling', 'reshaping' or 'retuning'.

Interference between DNA binding activities of AP-1 and GR transcription factors in rat thymocytes undergoing dexamethasone-induced apoptosis.

The early molecular events of glucocorticoid-induced apoptosis have been investigated by studying glucocorticoid receptor levels, as well as binding activities to GRE and AP-1 sequences, using nuclear extracts from dexamethasone (Dex)-treated rat thymocytes. When the time-course of glucocorticoid-receptor complexes in nuclei of thymocytes was evaluated by binding studies using the tritiated ligand, we found that nuclear accumulation of radioactive complexes occurred in the first hour of incubation, and was followed by a progressive decline. This trend was confirmed by immunoblotting of nuclear proteins using a monoclonal anti-glucocorticoid receptor antibody. When the kinetics of binding activity to AP-1 and GRE sequences were studied, using nuclear extracts prepared from Dex-treated thymocytes in gel shift assays, we found peaks at 1 and 2 h after Dex treatment, and a return to basal levels in the following hours. Binding specificity was proved by competition studies using non-radioactive sequences, including mutated AP-1. Unexpectedly, however, protein binding to GRE was better competed for by AP-1 sequence than by GRE itself. Data obtained using the super gel shift assay suggested that AP-1/Jun can be responsible for the high affinity for the GRE sequence. Thus, we report here for the first time that an interference between AP-1 and GR in the binding to DNA consensus sequences-previously described in other biological systems-also occurs during apoptosis induced by glucocorticoids in lymphoid cells.

Is polyamine decrease a common feature of apoptosis? Evidence from gamma rays- and heat shock-induced cell death.

Here we report that in rat thymocytes undergoing apoptosis upon two different stimuli, such as heat shock treatment and gamma irradiation, an early mRNA accumulation of ornithine decarboxylase (ODC)--the rate-limiting enzyme of polyamine biosynthesis--was followed by a very marked increase in ODC activity (28-40 and 6-8-fold, respectively). However, polyamine levels started to decrease before the appearance of DNA laddering, being putrescine and spermidine strongly diminished (8-12 hs), and spermine even depleted (12 hs). Taken together with our previous data on another model of apoptosis, i.e., glucocorticoid-induced cell death (Desiderio et al., Cell Growth Differ. 6: 505-513, 1995), these results suggest that an imbalance of polyamine metabolism, i.e., a strong activation of ODC and a paradoxical decrease of the intracellular polyamine content, might be a general feature of the apoptotic process.

Involvement of ornithine decarboxylase and polyamines in glucocorticoid-induced apoptosis of rat thymocytes.

Ornithine decarboxylase (ODC), the first and rate-limiting enzyme of polyamine metabolism, has been shown to be required for entry into and progression through the cell cycle. However, the role of ODC and polyamines in apoptosis remains to be determined. We have examined ODC expression and polyamine levels in thymocytes activated to undergo apoptosis by dexamethasone treatment. We have demonstrated a rapid and reversible induction of ODC (mRNA and activity), as previously reported for the mRNA expression of other "early" genes, c-fos, c-jun, and c-myc, in the same experimental model. Surprisingly, polyamine levels diminished progressively starting at 2-4 h after dexamethasone treatment, and spermine was depleted at 8-12 h. This seemed to be relevant since increasing the intracellular polyamine levels by exogenous spermine administration prevented the DNA "laddering" (2-4 h) and the DNA loss from the nucleus (8-18 h) due to dexamethasone treatment. Moreover, the activities of spermidine/spermine N1-acetyltransferase, which controls the cytosolic polyamine interconversion pathway, and of spermidine N8-acetyltransferase, which regulates the nuclear pool and functions of polyamines, were measured in apoptotic cells. Spermidine/spermine N1-acetyltransferase activity progressively increased and might be responsible for spermidine and spermine excretion as acetyl derivatives. In contrast, spermidine N8-acetyltransferase activity remained unchanged. A completely different scenario was observed in proliferating concanavalin A-treated thymocytes, studied for comparison. In this case, polyamine levels increased, remaining at high values until 12 h. This is likely a consequence of the rapid and prolonged induction of ODC (mRNA and activity), accompanied by that of spermidine/spermine N1-acetyltransferase (mRNA and activity).(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial modifications during rat thymocyte apoptosis: a study at the single cell level.

Apoptosis is an active type of cell death, occurring under several physiological and pathological conditions. The role of cellular organelles such as mitochondria in this process is still an open question. We recently described a new method to measure mitochondrial membrane potential in intact cells using flow cytometry. Using this method we studied alterations of mitochondrial membrane potential in a classical model of apoptosis, i.e., dexamethasone-treated rat thymocytes. Moreover, apoptosis induced by heat shock was also studied in the same cells. Mitochondria are functionally intact during the early phases of apoptosis, when DNA fragmentation occurs, whereas early alteration in their potential and mass takes place after DNA damage. According to flow-cytometric analysis, the presence of a hypodiploid peak, an index of nuclear DNA loss, predates depolarization of mitochondrial membrane and decrease of mitochondrial mass. Loss of plasma membrane integrity, which indicates cell death and is revealed by the permeability to propidium iodide, eventually follows. All these phenomena are more evident in dexamethasone-treated cells than in heat-shocked cells. Thus, in these types of apoptosis the involvement of mitochondria is apparently not a primary event.