Oncogene activation and tumor suppressor gene inactivation during multistage mouse skin carcinogenesis.

The mouse skin multistage model of carcinogenesis is an ideal system in which to study questions related to the timing of oncogene activation and inactivation of tumor suppressor genes. A number of laboratories have shown that an early event associated with chemical initiation of mouse skin tumors involves activation of the Harvey-ras oncogene. To approach the question of timing of loss of tumor suppressor genes in skin carcinogenesis, we have utilized a model system developed by Kulesz-Martin in which cloned mouse keratinocytes were initiated with DMBA and variant clones with benign or malignant phenotypes were developed. We have generated somatic cell hybrids between the parental clone and the variants to study the potential loss of tumor suppressor activity during the progression of cells from the initiated to benign and to the malignant phenotypes. Somatic cell hybrids generated between the parental, normal cell strain (i.e., 291) and a malignant cell variant (i.e., 05), that produces moderately differentiated squamous cell carcinomas (SCCs), failed to produce tumors indicating tumor suppressor activity in the 291 cells. The 291 cells and a benign papilloma producing variant (i.e., 09) were able to partially suppress in hybrids the tumorigenicity of another malignant cell line (i.e., 03) which produces poorly-differentiated SCCs. Suppression of 03 tumorigenicity by the benign tumor cell, 09, was less than that seen with the normal cell, 291. These results indicated two potentially different suppressor activities were inactivated during progression of normal 291 to malignant 03 cells. We have also obtained evidence that constitutive AP-1 activity plays a role in the maintenance of the malignant phenotype of SCC cell lines. Two different SCC cell lines, 308 10Gy5 and PDV, demonstrate constitutive AP-1 activity. To examine the role of this activity in malignant progression, we stably expressed a transactivation deletion mutant of the human c-jun gene in these cell lines. Expression of this mutant c-jun protein blocked transcriptional transactivation of AP-1 responsive reporter CAT constructs driven by jun, human collagenase, and the mouse stromelysin promoters. These malignant cells were not only inhibited in their AP-1 transactivation response, but also in their ability to form SCCs upon s.c. injection into athymic nude mice. These results support the idea that inhibition of AP-1-mediated transcriptional transactivation is in some cases sufficient to suppress the tumorigenic phenotype of malignant mouse epidermal cells.

Interaction of visual and non-visual signals in the initiation of smooth pursuit eye movements in primates.

The initiation of smooth pursuit eye movements (PEM) by visual and non-visual signals was analysed in humans and monkeys. While PEM latency ranged around 150 ms when a purely visual target was provided, it often dropped to about 0 ms, or even became negative, when target movement was coupled to the subject's arm; this suggests that signals about the intention to move the arm can be evaluated for PEM control. Eye movements always started in the visually correct direction, independent of the sign of coupling between arm and target; from this we conclude that intentional signals are not mere triggers, but also convey directional information. Short-latency PEM trials were intermixed with those characterized by normal latencies, which often resulted in bimodal latency distributions; this suggests that visual and intentional signals compete for the control of PEM.

Inhibitory mechanisms terminating paroxysmal depolarization shifts in hippocampal neurons of rats.

The mechanisms responsible for the termination of paroxysmal depolarization shifts (PDS) were studied with intracellular recordings on CA1 neurons of rat hippocampal slices. Epileptiform activity was induced by application of penicillin, bicuculline or Mg-free artificial cerebrospinal fluid (ACSF). PDS in penicillin-containing and Mg-free ACSF were markedly prolonged when GABAA-dependent IPSPs were blocked by bicuculline. PDS in bicuculline-containing ACSF were furthermore prolonged after block of potential dependent K+ conductances by TEA. TEA also exerted some effect on PDS induced by penicillin containing or Mg-free ACSF. Block of GABAB-dependent IPSPs or Ca(2+)-dependent K+ currents did not affect PDS duration in any of the three models. It is concluded that PDS termination is due to active inhibitory processes which comprise different components. If one of these components is blocked another inhibitory component governs PDS repolarization resulting in PDS with a slightly different duration but otherwise unchanged features.

Electrophysiological changes in the surrounding brain tissue of photochemically induced cortical infarcts in the rat.

Small infarctions in the parietal cortex of Wistar rats were produced photochemically using the Rose Bengal technique. The infarctions evoked reproducible cortical lesions of about 2 mm diameter. In the surrounding brain tissue changes in electrophysiological responses occurred. Whereas in control animals a paired-pulse inhibition could be evoked all over the neocortex, in infarcted animals the paired-pulse inhibition was significantly reduced or even absent within an area extending up to 5 mm lateral from the lesion center. The changes in paired-pulse inhibition were already present on the first day and persisted at least up to 60 days after infarction. These functional changes may contribute to neurological deficits occurring after cerebral infarcts.

Spatial distribution of fluorescence intensity within large droplets and its dependence on dye concentration.

The dependence of fluorescence intensity distributions within droplets on added dye concentration has been calculated by extension of the geometrical-optics approximation and verified by experimental observations. With rising dye concentration, surface plots of the equatorial fluorescence pattern show decreasing relevance of intensity enhancement at focusing points of internal light rays and increasing effects of linear absorption on the characteristic features of the distribution. For comparison with experimentally obtained images of the fluorescence intensity distribution within droplets, a method for calculating volume-integrated intensity distributions was developed in which image distortion at the fluid-air interface is included. A comparison of the calculated and the experimentally determined fluorescence intensity distributions within a droplet confirmed the accuracy of the geometrical-optics approach at high dye concentrations. However, discrepancies from experimental results are visible at low dye concentrations owing to nonlinear optical effects.

Lead reduces depolarization-induced calcium entry in cultured DRG neurons without crossing the cell membrane: fura-2 measurements.

1. Cultured dorsal root ganglion of rat pups were depolarized by exposure to 50 mM K+ and the rise of [Ca2+]i was measured using fura-2 as an indicator. 2. Lead in the extracellular solution reduced the rise of [Ca2+]i in a concentration-dependent manner, with a threshold concentration of 0.25 microM. More than 80% of the calcium entry was prevented by approximately 5 microM lead. The IC50 and the Hill coefficient were 3.1 microM and 1, respectively. 3. This effect was considered to be due to a reduction of VACCCs, since applications of NMDA did not result in any rise of [Ca2+]i. 4. Since Pb2+ itself changes the fura-2 signal in a typical and characteristic manner, fura-2 is also an indicator for Pb2+. No changes in fura-2 signals were detected when lead (5 microM) was applied for several minutes in the absence of calcium, indicating that Pb2+ did not enter the cells. 5. Thus it is concluded that lead prevents calcium entry by reducing VACCCs but does not cross the cell membrane itself.

Calcium-dependent potassium current following penicillin-induced epileptiform discharges in the hippocampal slice.

Penicillin-induced paroxysmal depolarization shifts (PDS) are followed by prolonged afterhyperpolarizations of about 2 seconds duration. Intracellular injection of EGTA blocked a late component of the afterhyperpolarizations; an early one lasting up to one second was only slightly reduced by EGTA. It is concluded that afterhyperpolarizations following penicillin-induced PDS comprise different components: an initial one lasting up to one second which is not Ca2+-dependent and a slow one lasting up to two seconds which is caused by a Ca2+-dependent K+ current.

Afterpotentials following penicillin-induced paroxysmal depolarizations in rat hippocampal CA1 pyramidal cells in vitro.

Epileptic discharges were induced by superfusion of rat hippocampal slices with penicillin. Under these conditions the neurons generated paroxysmal depolarization shifts (PDS) after electrical stimulation of Schaffer collaterals. The PDS were followed by large afterhyperpolarizations lasting about 2 s. The mechanisms causing these afterhyperpolarizations were studied in CA1 pyramidal cells. A late component of the afterhyperpolarizations, which determined their overall duration, was blocked by intracellular application of EGTA and reduced by superfusion with 8-Br-cAMP. In the same neurons these drugs had a comparable effect on afterhyperpolarizations following depolarizing current injections; it was therefore concluded that the late component of the PDS afterhyperpolarizations was caused by a slow Ca2(+)-activated K+ current. An initial fast component of PDS afterhyperpolarizations, which peaked about 60 ms after PDS onset, was reduced by EGTA but not affected by 8-Br-cAMP suggesting that the fast Ca2(+)-activated K+ current also contributed to the PDS afterhyperpolarizations. Superfusion of the slice with the gamma-aminobutyric acid B receptor (GABAB) antagonists phaclofen or 5-aminovalerate reduced the amplitude of the afterhyperpolarizations during the first 1000 ms but did not affect the late Ca2(+)-dependent component, indicating that a GABAB-mediated K+ inhibitory postsynaptic potential (IPSP) contributed to the PDS afterhyperpolarization. Intracellular injection of Cl- revealed that an early part of the afterhyperpolarizations lasting about 500 ms was Cl(-)-dependent. This component was blocked by superfusion of the slices with bicuculline, suggesting that a GABAA-mediated Cl- IPSP contributed to the PDS afterhyperpolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Participation of interneurons in penicillin-induced epileptic discharges.

Interneurons of rat motor cortex in vivo and of rat hippocampal slices were studied during penicillin-induced epileptic discharges. Synchronous with pyramidal cells, they showed transient depolarizations similar to paroxysmal depolarization shifts in pyramidal cells. The transient depolarizations were followed by hyperpolarizing or depolarizing afterpotentials lasting 600 to 1200 ms. During the transient depolarizations and the afterdepolarizations the interneurons discharged with increased frequency. This may contribute to the enlarged and prolonged synaptic inhibitions following interictal discharges in pyramidal cells.

Inhibitory mechanisms in epileptiform activity induced by low magnesium.

In rat hippocampal slices epileptiform activity was induced by superfusion with Mg(2+)-free artificial cerebrospinal fluid (ACSF). Paroxysmal depolarization shifts (PDS) were evoked by electrical stimulation of Schaffer collaterals. To investigate the afterpotentials that follow PDS, intracellular recordings were made from CA1 pyramidal cells. The experiments revealed that several components are engaged in the generation of PDS afterpotentials in Mg(2+)-free ACSF. A long lasting component which determined the overall duration of the PDS afterhyperpolarization was blocked by intracellular application of ethylenebis(oxonitrilo)-tetraacetate (EGTA); concomitantly, the afterhyperpolarizations following depolarizing current injections were blocked. This indicated that the long lasting component was due to a slow Ca(2+)-activated K+ current. The block of Ca(2+)-activated K+ current uncovered a depolarizing PDS afterpotential with an N-shaped voltage dependence, suggesting that this depolarizing afterpotential component may be due to an N-methyl D-aspartate (NMDA) conductance. Intracellular injection of Cl- revealed that the PDS were followed by Cl- currents lasting about 500 ms. This component could be blocked by application of bicuculline suggesting that it is due to a synaptically GABA-mediated (i.e. gamma-aminobutyric acid) Cl- current. A comparison of PDS afterpotentials in Mg(2+)-free ACSF and those in other models of epileptiform activity suggests that similar sequences of inhibitory components are activated in spite of different pharmacological alterations of membrane conductances which induce the epileptiform discharges.