Retinoblastoma family proteins as key targets of the small DNA virus oncoproteins.

RB, the most investigated tumor suppressor gene, is the founder of the RB family of growth/tumor suppressors, which comprises also p107 (RBL1) and Rb2/p130 (RBL2). The protein products of these genes, pRb, p107 and pRb2/p130, respectively, are also known as 'pocket proteins', because they share a 'pocket' domain responsible for most of the functional interactions characterizing the activity of this family of cellular factors. The interest in these genes and proteins springs essentially from their ability to regulate negatively cell cycle processes and for their ability to slow down or abrogate neoplastic growth. The pocket domain of the RB family proteins is dramatically hampered in its functions by the interference of a number of proteins produced by the small DNA viruses. In the last two decades, the 'viral hypothesis' of cancer has received a considerable renewed impulse from the notion that small DNA viruses, such as Adenovirus, Human papillomavirus (HPV) and Polyomavirus, produce factors that can physically interact with major cellular regulators and alter their function. These viral proteins (oncoproteins) act as multifaceted molecular devices that have evolved to perform very specific tasks. Owing to these features, viral oncoproteins have been widely employed as invaluable experimental tools for the identification of several key families of regulators, particularly of the cell cycle homeostasis. Adenovirus early-region 1A (E1A) is the most widely investigated small DNA tumor virus oncoprotein, but relevant interest in human oncology is raised by the E1A-related E7 protein from transforming HPV strains and by Polyomavirus oncoproteins, particularly large and small T antigens from Simian virus 40, JC virus and BK virus.

Human HLA-DR alpha gene: a rare oligonucleotide (GTATA) identifies an upstream sequence required for nuclear protein binding.

Synthetic oligonucleotides containing putative regulatory sequences are currently employed to identify and isolate genes coding for nuclear binding factors. Upstream DNA sequences of eukaryotic genes required for transcriptional activity and tissue specificity can be identified by means of biochemical techniques as well as computer analysis using homology searching. An alternative approach has been recently proposed by our research group. Scanning DNA sequences 1.8 megabases in length from a Genetic Sequence Data Bank, we have identified rare oligonucleotides 5 base pairs (bp) long, which are localized within or close to regulatory segments in mammalian promoters. In this paper we demonstrate that the rare GTATA sequence identifies an upstream region of the HLA-DR alpha gene which operates in conjunction with the sequence AGAAGTCAG, homologous to a box found in many interferon-inducible genes, in binding nuclear proteins.

CXC chemokines interleukin-8 (IL-8) and growth-related gene product alpha (GROalpha) modulate Purkinje neuron activity in mouse cerebellum.

We give here evidence that Purkinje neurons (PNs) of mouse cerebellar slices studied with patch clamp technique combined with laser confocal microscopy, respond to human IL-8 and GROalpha by (i) a cytosolic Ca2+ transient compatible with inositol (1,4,5) trisphosphate (InsP3) formation; (ii) an enhancement of the neurotransmitter release; and (iii) an impairment of the long-term depression of synaptic strength (LTD). It was also found the expression of IL-8 receptor type 2 in PN and granule cells by immunofluorescence, immunoblotting and RT-PCR analysis. Considered together these findings suggest that IL-8 and GROalpha may play a neuromodulatory role on mouse cerebellum.

Modulation of the neurotransmitter release in rat cerebellar neurons by GRO beta.

We report here that, in cultured cerebellar granule cells, the CXC chemokine GRObeta stimulates the signaling pathway of the extracellular signal-regulated kinases, and enhances both evoked and spontaneous postsynaptic currents in patch clamped Purkinje neurons from rat cerebellar slices. The GRObeta-induced enhancement of the excitatory post-synaptic currents evoked by stimulating the parallel fibres is blocked by the inhibitor of the extracellular signal-regulated kinases pathway PD98059, which also reduces both basal frequency of spontaneous post-synaptic currents and mean amplitude of evoked excitatory post-synaptic currents. Our results suggest that GRObeta modulates neurotransmitter release in the cerebellum through the activation of the extracellular signal-regulated kinases pathway.

Alpha 5 subunit forms functional alpha 3 beta 4 alpha 5 nAChRs in transfected human cells.

nAChRs heterologously expressed in human cells after transient transfection with alpha 3 beta 4 alpha 5 or alpha 3 beta 4 subunit cDNAs exhibited similar sensitivities to antagonists and comparable functional channel profiles. However, the sum of two Hill equations was required for best fitting the ACh dose-current response curves after co-expression of alpha 5, alpha 3 and beta 4 subunits. One component was comparable to that obtained in alpha 3 beta 4-transfected cells, while the additional component, putatively attributed to an alpha 3 beta 4 alpha 5 nAChR population, showed a Hill coefficient > 2 and a nine-fold greater half-maximal ACh concentration (EC50). These results suggest that the alpha 5 subunit participates in the assembly of alpha 3 beta 4 alpha 5 nAChRs complexes in human cells, adding a new member to the family of neuronal nicotinic receptors.

Phorbol ester modulation of both delta-mutant and subunit-omitted nicotinic receptors expressed in Xenopus oocytes.

The action of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the potent stimulator of protein kinase C (PKC), on acetylcholine-activated currents (I(Ach)) was investigated in voltage clamped Xenopus laevis oocytes injected with RNAs encoding murine embryonic nicotinic acetylcholine receptor (AChR) subunits. Comparable potentiation and acceleration of decay of I(ACh) were observed within minutes of phorbol ester application in oocytes injected with various RNA subunit combinations: (i) alpha beta gamma delta; (ii) alpha beta gamma; (iii) alpha beta delta; and (iv) alpha beta gamma delta(AAA), a mutant of the delta subunit with serine residues 360-361-362 mutated to alanine. Our findings indicate that the effects on I(ACh) induced by PKC stimulation are independent of both gamma and delta subunits and, accordingly, of the presence of PKC phosphorylation sites on delta subunit. It is here suggested a novel PKC-dependent modulatory mechanism of cholinergic receptor which does not involve direct phosphorylation of the AChR and requires phosphorylation of intermediate regulatory protein(s).

TNF-alpha increases the frequency of spontaneous miniature synaptic currents in cultured rat hippocampal neurons.

Tumor necrosis factor-alpha (TNF-alpha) is a cytokine secreted by activated astrocytes and is known to alter evoked synaptic activity in slices of adult rat hippocampus. In this paper we show that TNF-alpha increases the frequency of spontaneous miniature synaptic currents in cultured hippocampal neurons, acting at nanomolar concentrations. In addition, we show that the mRNA for the 55 kDa TNF-alpha receptor (TNF-R1) is detected in embryonic rat hippocampal cultures, as well as in acutely dissected embryonic and adult rat hippocampi. Possible transduction pathways mediating the TNF-alpha effect are discussed.

Identification of a 66 kD heat shock protein (HSP) induced in M-14 human melanoma cells by severe hyperthermic treatment.

Following severe hyperthermic treatment M-14 cells synthesize at high rate a new protein of about 66 kD, in addition to the three well known major HSPs (HSP 28, HSP 70 and HSP 90). This 66 kD protein is constitutively expressed at low levels and its rate of synthesis is not enhanced by mild hyperthermic exposures (40 degrees C for 2-4 h; 42 degrees C for 1-3 h), sufficient to induce the three major HSPs. The 66 kD protein is induced whenever the thermal dose administered to cells attains a threshold, roughly corresponding to a 50% reduction in survival. The 66 kDa protein is not induced by a variety of compounds (disulfiram, arsenate, cadmium) able to elicit a stress response in M-14 cells, as indicated by enhanced synthesis of the three major HSPs. Once induced by a treatment at 45 degrees C for 15 min, the rate of synthesis of the 66 kD protein remains above the control level for 16-20 h during recovery from the stress, while the synthesis of HSP 70 is shut off between 8 and 12 h. Immunoblotting and immunoprecipitation studies showed that the 66 kD protein shares immunological determinant(s) with HSP 70. Pulse-chase experiments demonstrated that the 66 kD protein is not a degradation product or a late post-transcriptional modification of HSP 70. It is proposed that the 66 kD protein is a previously unrecognized heat shock protein (HSP 66), characterized by an unusually high threshold for its induction.

Stress response, survival and enhancement of heat sensitivity in a human melanoma cell line treated with L-canavanine.

L-Canavanine, like other aminoacid analogs, induces the synthesis of heat shock proteins (HPSs) but, unlike heat or other stressing agents, it fails to induce thermotolerance. We have studied the synthesis and the intracellular distribution of HSPs induced by canavanine, the effects of this analog on the viability and thermal sensitivity of a human melanoma cell line (M14) and the capacity of canavanine-induced HSPs to self regulate their own synthesis. Evidence indicates that the HSP induction is time--and dose--dependent and, also in the presence of arginine, is not associated with the development of thermotolerance. On the contrary, cells become more heat sensitive and are less efficient in the control of the feed-back mechanism that regulates HSP synthesis. The possible utilization of this substance as a potential aid for the treatment of tumors, in association with heat, was examined.

Selective over-expression of mRNA coding for 90 KDa stress-protein in human ovarian cancer.

The mRNA expression of the KDa stress-protein (SP) was assayed in normal and neoplastic human ovary. It was found that the expression level is low in normal ovary and in benign tumors and increases in the more advanced stages of ovarian cancer. A low level of mRNA coding for the 70 KDa SP--the most prominent among the inducible SPs-was constantly found in all specimens. No relationship was observed between SP 90 mRNA abundance and the receptor status of epidermal growth factor, estrogen and progesterone.

Preliminary evaluation of HER-2/neu oncogene and epidermal growth factor receptor expression in normal and neoplastic human ovaries.

The HER-2/neu oncogene (a member of the Erb-like oncogene family) is distinct from but closely related to the c-erb B gene which encodes the epidermal growth factor receptor (EGFr). HER-2/neu gene amplification was found in a large number of mammary carcinomas and there was a strong correlation between this phenomenon and poor prognosis. In our study HER-2/neu oncogene expression was determined in 16 malignant ovarian tumors, 2 ovarian lymphomas and 5 normal ovaries. The HER-2/neu gene was found both in normal ovaries and malignant tumors, without any apparent difference among the various histological types. In all the specimens examined, HER-2/neu expression did not seem to be related to EGF binding capacity.

Preliminary evaluation of HER-2/neu oncogene and epidermal growth factor receptor expression in normal and neoplastic human ovaries.

The HER-2/neu oncogene (a member of the Erb-like oncogene family) is distinct from but closely related to the c-erb B gene which encodes the epidermal growth factor receptor (EGFr). HER-2/neu gene amplification was found in a large number of mammary carcinomas and there was a strong correlation between this phenomenon and poor prognosis. In our study HER-2/neu oncogene expression was determined in 16 malignant ovarian tumors, 2 ovarian lymphomas and 5 normal ovaries. The HER-2/neu gene was found both in normal ovaries and malignant tumors, without any apparent difference among the various histological types. In all the specimens examined, HER-2/neu expression did not seem to be related to EGF binding capacity.

Induction of stress proteins in murine and human melanoma cell cultures.

The induction of stress proteins was studied in two human and two murine melanoma cell lines. Exposure for 1 h to heat (42 degrees C), to ethanol (6%), to arsenate (100 microM) and to disulfiram (50 microM) induced the expression of SPs with apparent molecular weights of 100, 86, 70-72 and 24-26 Kd. Quantitation of the single SPs indicated that the basal level as well as the enhanced synthesis following the various stressors were different in each cell line. The induction of the 100 Kd species occurred in only one murine melanoma and not in the others. The 86 and in particular the 70-72 Kd species were the most prominent groups, whereas the 24-26 SPs were induced only following arsenate and disulfiram exposure in the three melanoma cell lines. In one of the murine melanomas, the expression of SPs was markedly reduced compared to the other cell lines. No definite specific patterns of SP expression could be identified in tumors of the same histologic type.

Absence of acquired thermotolerance in murine tumors unable to increase the expression of heat shock proteins following stress stimuli.

The induction of thermotolerance was studied in two groups of murine tumors, one able to produce heat shock proteins (HSP) and the other entirely lacking HSP expression in response to various stress inducers. Heat treatments were performed in vitro and the development of thermotolerance was then evaluated in vivo. The data obtained on the death rate of mice inoculated with tumor cells previously conditioned at 42 degrees C for 1 h and then challenged at 45 degrees C for 30 min following 2 h of reincubation at 37 degrees C, show that the rate of survival is far higher in mice inoculated with HSP negative tumor cells. This indicates that a large number of cells able to increase HSP synthesis following stress escape heat killing, whereas cells unable to express HSP after adequate stimuli are less tolerant against heat challenge.

Sgk1 enhances RANBP1 transcript levels and decreases taxol sensitivity in RKO colon carcinoma cells.

The serum- and glucocorticoid-regulated kinase (Sgk1) is essential for hormonal regulation of epithelial sodium channel-mediated sodium transport and is involved in the transduction of growth factor-dependent cell survival and proliferation signals. Growing evidence now points to Sgk1 as a key element in the development and/or progression of human cancer. To gain insight into the mechanisms through which Sgk1 regulates cell proliferation, we adopted a proteomic approach to identify up- or downregulated proteins after Sgk1-specific RNA silencing. Among several proteins, the abundance of which was found to be up- or downregulated upon Sgk1 silencing, we focused our attention of RAN-binding protein 1 (RANBP1), a major effector of the GTPase RAN. We report that Sgk1-dependent regulation of RANBP1 has functional consequences on both mitotic microtubule activity and taxol sensitivity of cancer cells.

Protein interactions provide new insight into Nm23/nucleoside diphosphate kinase functions.

Nm23-NDPKs besides contributing to the maintenance of the cellular nucleoside triphosphate pool, exert regulatory properties in a variety of cellular events including proliferation, invasiveness, development, differentiation, and gene regulation. This review focuses on recently discovered protein-protein interactions involving the Nm23 proteins. The findings herein summarized provide new and intriguing suggestions for a more extensive understanding of the biological functions of the Nm23 proteins.

The desensitization of the embryonic mouse muscle acetylcholine receptor depends on the cellular environment.

The rate of desensitization of nicotinic acetylcholine (ACh) receptor (nAChR), an important characteristic of nAChR function, was studied in myotubes of the mouse C2C12 cell line at different times after fusion, by measuring the decay of ACh-evoked currents (IACh) under various patch-clamp configurations. We observed a progressive slowing of IACh decay rate (half-decay time rose from about 0.5 s to over 5 s) in myotubes of increasing size (i.e. age) under all experimental conditions, except in outside-out patches, when IACh decayed as fast as in the smallest myotubes. Single-channel conductance (about 35 pS) and open time (about 3.5 ms), measured in outside-out and cell-attached patches, were independent of myotube size. In Xenopus oocytes injected with poly(A+)RNA extracted from C2C12 myoblasts or mature myotubes, IACh decay was about 50 times slower than in myotubes. Neither cAMP-dependent nor diacylglycerol-dependent protein kinases, actin nor microtubule polymerization state influenced IACh decay. Our data indicate that the cellular environment, but not readily dialysable cytosolic factors, markedly influences the functional behaviour of nAChR.

Characterization of the heat shock response in M-14 human melanoma cells continuously exposed to supranormal temperatures.

The heat shock response elicited in a human melanoma cell line (M-14) by continuous exposures to supranormal temperatures has been characterized. The electrophoretic patterns of polypeptides labeled in vivo at different time-intervals during a continuous heating at 42 degrees C show that the hyperthermic stress induces the synthesis of three HSPs, with molecular weights, respectively, of 86 kDa, 70-72 kDa and 26 kDa. The relative rate of synthesis of the 70-72 kDa HSP--the preeminent HSP--increases during the first hours of treatment, reaching the maximum value after about 9 hr. Later on, the rate of synthesis of this protein progressively decreases, finally attaining a steady state level only slightly exceeding the constitutive one. On the contrary, the smaller molecular weight HSP is synthesized at an apparently constant rate in the course of 21 hr of heating treatment. A continuous exposure at 40 degrees C induces the synthesis of the same three HSPs observed in cells heated at 42 degrees C, but the rate of synthesis of all these HSPs is not so greatly enhanced over the control values as in the 42 degrees C-heated cells. Moreover, the repression of the 70-72 kDa HSP synthesis is faster, taking place within 4-6 hr of treatment. Coomassie blue stained gels show that a polypeptide, coincident with the 70-72 kDa HSP, accumulates in the course of a continuous heating either at 42 degrees C and at 40 degrees C. The final intracellular level attained by this protein species results higher in 42 degrees C-treated cells than in 40 degrees C-treated ones. Hybridization experiments between total RNAs obtained from cells heated at 42 degrees C and a radioactive DNA probe (containing sequences complementary to the mRNA coding for the human 70 kDa HSP) demonstrate that the kinetics of accumulation and decay of the 70 kDa HSP-mRNAs correlate with the kinetics of induction and repression of the corresponding protein.

Threonine-for-leucine mutation within domain M2 of the neuronal alpha(7) nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist.

A study was made of the effects of 5-hydroxytryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken alpha(7) subunit. Acetylcholine (AcCho) elicited large currents (IAcCho) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC50) of 56 microM and a Hill coefficient (nH) of 1.2. The inhibition of IAcCho by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism. 5HT alone did not elicit membrane currents in oocytes injected with the wild-type alpha(7) subunit cDNA. In contrast, 5HT elicited membrane currents (I5HT) in oocytes injected with cDNA encoding an alpha(7) mutant subunit with a threonine-for-leucine-247 substitution (L247T alpha(7)). I5HT was inhibited by the potent nicotinic receptor blockers alpha-bungarotoxin (100 nM) and methyllycaconitine (1 microM). Furthermore, the characteristics of I5HT, including its voltage dependence, were similar to those of IAcCho. The 5HT dose-I5HT response gave an apparent dissociation constant EC50 of 23.5 microM and a Hill coefficient nH of 1.7, which were not modified by the presence of AcCho. Similarly, the apparent affinity of L247T alpha(7) for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal alpha(7) nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.