Comparative studies of intracellular Ca2+ in strongly and weakly metastatic rat prostate cancer cell lines.

The metastatic ability of prostate cancer cells involves differential expression of ionic mechanisms. In the present study, using electrophysiological recordings and intracellular Ca2+ measurements, we investigated Ca2+ related signalling in two rat prostate cancer (MAT-LyLu and AT-2) cell lines of markedly different metastatic potential. Whole-cell voltage clamp experiments indicated the absence of an inward current carried through voltage-dependent Ca2+ channels in either cell line. A Ca2+-dependent component was also absent in the voltage-activated outward K+ currents. Indo-1 microfluorimetry confirmed these results and also revealed marked differences in the resting level of intracellular Ca2+ and the ability of the two cell lines to regulate intracellular Ca2+. The weakly metastatic AT-2 cells displayed a significantly higher resting intracellular Ca2+ than the related but strongly metastatic MAT-LyLu cell line. Increasing extracellular K+ decreased intracellular Ca2+ in the AT-2 but had no effect on intracellular Ca2+ levels in the MAT-LyLu cells. Furthermore, increasing extracellular Ca2+ increased intracellular Ca2+ in AT-2 but, again, had no effect on MAT-LyLu cells. These results suggested the presence of a tonic, voltage-independent Ca2+ permeation mechanism operating specifically in the AT-2 cells. The influx of Ca2+ into the AT-2 cells was suppressed by both CdCl2 (100-300 microM) and SKF-96365 (10-30 microM). It is concluded that the strongly metastatic MAT-LyLu cell line lacks a voltage-independent basal Ca2+ influx mechanism that is present in the weakly metastatic AT-2 cells.

Differential expression of voltage-activated Na+ currents in two prostatic tumour cell lines: contribution to invasiveness in vitro.

The voltage-gated ionic currents of two rodent prostatic cancer cell lines were investigated using the whole-cell patch clamp technique. The highly metastatic Mat-Ly-Lu cells expressed a transient, inward Na+ current (blocked by 600 nM tetrodotoxin), which was not found in any of the weakly metastatic AT-2 cells. Although both cell lines expressed a sustained, outward K+ current, this occurred at a significantly higher density in the AT-2 than in the Mat-Ly-Lu cells. Incubation of the Mat-Ly-Lu cell line with 600 nM tetrodotoxin significantly reduced the invasive capacity of the cells in vitro. Under identical conditions, tetrodotoxin had no effect on the invasiveness of the AT-2 cells.

The influence of drugs and disease activity on biochemical and haematological data in rheumatoid arthritis.

Biochemical and haematological data from 218 patients with rheumatoid arthritis were analysed and compared with data from a reference hospital population. The comparison demonstrated significant differences in several biochemical and haematological tests and that the patterns of change are different between males and females. The data were also analysed by conventional statistical methods and discriminant analysis using a computer to establish which tests were most influenced by the activity of the disease and drug therapy. The results obtained demonstrate marked difference between groups of patients with different disease activity or receiving different drugs. The discriminant analysis also identified those tests which differentiate these groups most effectively.

Response-independent milk delivery enhances persistence of pellet-reinforced lever pressing by rats.

If, during training, one stimulus is correlated with a higher rate of reinforcement than another, responding will be more resistant to extinction in the presence of that higher rate signal, even if many of the reinforcers have been presented independently of responding. For the present study we asked if the response-independent reinforcers must be the same as the response-dependent reinforcers to enhance the response's persistence. Twelve Long-Evans hooded rats obtained 45-mg food pellets by lever pressing (variable-interval 100-s schedules) in the presence of two discriminative stimuli (blinking vs. steady lights) that alternated every minute during daily sessions. Also, in the presence of one of the stimuli (counterbalanced across rats), the rats received additional response-independent deliveries of sweetened condensed milk (a variable-time schedule). Extinction sessions were exactly like training sessions except that neither pellets nor milk were presented. Lever pressing was more resistant to extinction in the presence of the milk-correlated stimulus when (a) the size of the milk deliveries during training (under a variable-time 30 s schedule) was 0.04 ml (vs. 0.01 ml) and (b) 120-s or 240-s blackouts separated components. Response-independent reinforcers do not have to be the same as the response-dependent reinforcers to enhance persistence.

Response rate viewed as engagement bouts: effects of relative reinforcement and schedule type.

The rate of a reinforced response is conceptualized as a composite of engagement bouts (visits) and responding during visits. Part I of this paper describes a method for estimating the rate of visit initiations and the average number of responses per visit from log survivor plots: the proportion) of interresponse times (IRTs) longer than some elapsed time (log scale) plotted as a function of elapsed time. In Part 2 the method is applied to IRT distributions from rats that obtained food pellets by nose poking a lighted key under various multiple schedules of reinforcement. As expected, total response rate increased as a function of (a) increasing the rate of reinforcement (i.e., variable-interval [VI] 4 min vs. VI 1 mi), (b) increasing the amount of the reinforcer (one food pellet vs. four pellets), (c) increasing the percentage of reinforcers that were contingent on nose poking (25% vs. 100%), and (d) requiring additional responses after the end of the VI schedule (i.e., adding a tandem variable-ratio [VR] 9 requirement). The first three of these variables (relative reinforcement) increased the visit-initiation rate. The tandem VR, in contrast, increased the number of responses per visit. Thus, variables that have similar effects on total response rate can be differentiated based on their effects on the componemts of response rate.

Incidence and risk factors for development of malignant neoplasia after feline renal transplantation and cyclosporine-based immunosuppression.

The objective of this retrospective study is to report and analyse the incidence of and risk factors for post-transplant malignant neoplasia (PTMN) in feline renal transplant recipients (cases, n = 45) and compare incidence to a population of cats that did not receive a transplant (controls, n = 79). Information from the medical records of cases regarding signalment, blood work and concomitant disease, post operative cyclosporine concentrations, survival time (ST), and whether PTMN developed, the type of PTMN, time to occurrence (TTO), and ST after diagnosis was gathered. PTMN occurred in 11 of 45 (24%) cases, of which, four were lymphoma. Median TTO of all PTMN was 1020 days. Median TTO of lymphoma was 454 days. Median ST after diagnosis of PTMN was 15 days. No risk factors were identified. Compared with control cats, cases had more than six times higher odds of developing PTMN compared with control cats (P < 0.01).

Electrophysiological characterization of voltage-gated Na+ current expressed in the highly metastatic Mat-LyLu cell line of rat prostate cancer.

Voltage-gated Na+ channels, classically associated with impulse conduction in excitable tissues, are also found in a variety of epithelial cell types where their possible functions are not known so well. We have previously reported expression of a voltage-gated Na+ channel specifically in the highly metastatic Mat-LyLu rat prostate cancer cell line; blockage of the current with tetrodotoxin (TTX) significantly reduced the invasiveness of the cells in vitro, suggesting that the channel may have a functional role in metastasis. The aim of the present study was to characterize this current using the whole-cell patch clamp recording technique, and compare it to Na+ currents found in various other tissues. The inward current of the Mat-LyLu cells was abolished completely, but reversibly, in Na+-free solution, confirming that Na+ was indeed the permeant ion. Activation occurred at -40 mV and currents reached a maximal amplitude at around 6 mV. Boltzmann fits to current activation and steady-state inactivation revealed that the currents were half activated at about -15 mV and half inactivated at -80 mV. Both current inactivation and recovery from inactivation followed a double-exponential time course with fast and slow components. The Na+ currents were highly sensitive to block by TTX (IC50 approximately 18 nM), whilst 1 microM mu-conotoxin GIIIA mostly had no effect. 100 microM Cd2+ also had no effect on the current, whilst 2.5 mM Cd2+, Mn2+, and Co2+ each caused a depolarizing shift in activation and a reduction in peak conductance of around 20%. In conclusion, the Na+ channel expressed in the highly metastatic Mat-LyLu cell line appeared to have electrophysiological and pharmacological properties of TTX-sensitive channels. Further work is needed, however, to elucidate the exact nature of the channel protein and the mechanism(s) of its involvement in cellular invasiveness.

Effects of voltage-gated ion channel modulators on rat prostatic cancer cell proliferation: comparison of strongly and weakly metastatic cell lines.

BACKGROUND: The strongly metastatic MAT-LyLu and the weakly metastatic AT-2 rat prostatic cancer cell lines have been shown to express voltage-gated ion channels differentially. In the present study, the possible contribution of voltage-gated ion channel activity to the proliferation of these cell lines was investigated, in a comparative approach. METHODS: Several voltage-gated ion channel modulators were tested for their effects on proliferation over 54 hr, using an in vitro assay. The modes of action of the chemicals were monitored by electrophysiological (patch-clamp) recording. RESULTS: The voltage-gated K(+) channel blockers 4-aminopyridine (4-AP; 2 mM), margatoxin (5 nM), charybdotoxin (4.5 nM), and verapamil (50 microM) inhibited the K(+) channels of both cell lines by between 38-65% and reduced the proliferation of the AT-2 cell line, in a dose-dependent manner, by 8-51%. However, only 4-AP reduced proliferation of the MAT-LyLu cell line. Tetrodotoxin (6 microM) blocked completely the voltage-gated Na(+) channel expressed selectively in the MAT-LyLu cell line, but had no effect on the proliferation of either cell line. On the other hand, the presumed Na(+) channel "opener" veratridine (10-50 microM) reduced significantly, in a dose-dependent manner, the proliferation of both cell lines by up to approximately 30%. CONCLUSIONS: We conclude that the mechanism(s) controlling the proliferation of the weakly metastatic AT-2 cells involves voltage-gated K(+) channels. In contrast, the proliferation of strongly metastatic MAT-LyLu cells is much less dependent upon voltage-gated K(+) channel activity.

Expression and functional analysis of voltage-activated Na+ channels in human prostate cancer cell lines and their contribution to invasion in vitro.

Ion channels are important for many cellular functions and disease states including cystic fibrosis and multidrug resistance. Previous work in the Dunning rat model of prostate cancer has suggested a relationship between voltage-activated Na+ channels (VASCs) and the invasive phenotype in vitro. The objectives of this study were to 1) evaluate the expression of VASCs in the LNCaP and PC-3 human prostate cancer cell lines by Western blotting, flow cytometry, and whole-cell patch clamping, 2) determine their role in invasion in vitro using modified Boyden chambers with and without a specific blocker of VASCs (tetrodotoxin). A 260-kd protein representing VASCs was found only in the PC-3 cell line, and these were shown to be membrane expressed on flow cytometry. Patch clamping studies indicated that functional VASCs were present in 10% of PC-3 cells and blocking these by tetrodotoxin (600 nmol/L) reduced their invasiveness by 31% (P = 0.02) without affecting the invasiveness of the LNCaP cells. These results indicate that the reduction of invasion is a direct result of VASC blockade and not a nonspecific action of the drug. This is the first report of VASCs in a human prostatic cell line. VASCs are present in PC-3 but not LNCaP cells as determined by both protein and functional studies. Tetrodotoxin reduced the invasiveness of PC-3 but not LNCaP cells, and these data suggest that ion channels may play an important functional role in tumor invasion.

Predominant expression of Kv1.3 voltage-gated K+ channel subunit in rat prostate cancer cell lines: electrophysiological, pharmacological and molecular characterisation.

Voltage-gated K+ currents expressed in two rat prostate cancer ("Dunning") cell lines of markedly different metastatic ability were characterised using electrophysiological, pharmacological and molecular approaches. Whole-cell patch-clamp recordings showed that both strongly metastatic MAT-LyLu and weakly metastatic AT-2 cell lines possessed outward (delayed-rectifier type) K+ currents, which activated at around -40 mV. From the parameters measured, several characteristics of the two cell lines were similar. However, a number of statistically significant differences were noted for MAT-LyLu versus the AT-2 cells as follows: (1) current densities were smaller; (2) the slope factor for channel activation was smaller; (3) the voltage at which current was half-inactivated, and the slope factor for channel inactivation were greater; (4) the time constants for current decay at -20 and 0 mV were smaller; and (5) the residual peak current was larger following 60 s of repetitive voltage pulses for stimulation frequencies in the range 0.05-0.2 Hz. On the other hand, the K+ currents in both cell lines showed similar pharmacological profiles. Thus, the currents were blocked by 4-aminopyridine, tetraethylammonium, verapamil, margatoxin, and charybdotoxin, with highly similar IC(50)s for given blockers. The electrophysiological and pharmacological data taken together suggested expression of voltage-gated K+ channels of the Kv1 family, expression of the Kv1.3 subunit being predominant. Western blot and RT-PCR tests both confirmed that the cells indeed expressed Kv1.3 and to a lesser extent Kv1.4 and Kv1.6 channel alpha-subunits. In view of the similarity of channel expression in the two cell lines, voltage-gated K+ channel activity may not be a primary determinant of metastatic potential in the rat model of prostate cancer, but the possible contribution of K+ channel activity to the metastatic process is discussed.

The co-repressor mSin3A is a functional component of the REST-CoREST repressor complex.

The repressor REST/NRSF restricts expression of a large set of genes to neurons by suppressing their expression in non-neural tissues. We find that REST repression involves two distinct repressor proteins. One of these, CoREST, interacts with the COOH-terminal repressor domain of REST (Andres, M. E., Burger, C., Peral-Rubio, M. J., Battaglioli, E., Anderson, M. E., Grimes, J., Dallmanm J., Ballas, N. , and Mandel, G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9873-9878). Here we show that the co-repressor mSin3A also interacts with REST. The REST-mSin3A association involves the NH(2)-terminal repressor domain of REST and the paired amphipathic helix 2 domain of mSin3A. REST forms complexes with endogenous mSin3A in mammalian cells, and both mSin3A and CoREST interact with REST in intact mammalian cells. REST repression is blocked in yeast lacking Sin3 and rescued in its presence. In mammalian cells, repression by REST is reduced when binding to mSin3A is inhibited. In mouse embryos, the distribution of mSin3A and REST transcripts is largely coincident. The pattern of CoREST gene expression is more restricted, suggesting that mSin3A is required constitutively for REST repression, whereas CoREST is recruited for more specialized repressor functions.