Cyclin D1 Gene Amplification in Oral Squamous Cell Carcinoma using Differential Polymerase Chain Reaction

Neoplastic growth is characterized by alterations of oncogenes and antioncogenes. The interaction between activated oncogenes and functional deletion of antioncogene appears to be the driving force directing normal cells to uncontrolled growth resulting in tumor. In addition to those genes mentioned, other genes controlling the entry of cells into the cell cycle have recently been implicated in cancer development. The overexpression of the cyclin D1 gene, which has been mapped to 11q13, either by gene rearrangement or amplification has been noted in various malignant tumors. The product of the cyclin D1 gene forms a complex with cyclin-dependent protein kinases(CDK4) that governs a key transition in the cell cycle. The relationships between the overexpression of cyclin D1 assessed by immunihistochemistry and the amplification of the cyclin D1 gene by differential polymerase chain reaction(DPCR) using primers for dopamin D2 receptor gene in 13 cases of squamous cell carcinomas of the oral cavity have been studied. The semiquantitative assay of cyclin D1 amplification has been made by cyclin D1/dopamin D2 receptor(CD/DR) ratio. The results were as follows; 1. In the normal tissue and the tumor, the CD/DR ratios were 0.82 and 1.36 respectively. This implicates 1.65-fold amplification of cyclin D1 gene in tumor compared to that in normal tissue. 2. The tumor tissue which showed overexpression of cyclin D1 by immunohistochemistry revealed 2-fold amplification of cyclin D1 compared to the normal tissue. 3. The tumor tissue which showed mild expression of cyclin D1 by immunihistochemistry revealed 1.7-fold amplification of cyclin D compared to the normal tissue. 4. The cyclin D1 was overexpressed in the tumor tissue at the rate of 38%. Above results suggest that cyclin D1 has close correlation with the development of carcinoma in the oral cavity. But further studies were needed to elucidate the carcinogeneic mechanisms by comparative studies among cyclin D1, pRb and p53.

Magnesium suppresses the responses of dorsal horn cell to noxious stimuli in the rat

Magnesium ion is known to selectively block the N-methyl-D-aspartate (NMDA)-induced responses and to have anticonvulsive action, neuroprotective effect and antinociceptive action in the behavioral test. In this study, we investigated the effect of Mg2+ on the responses of dorsal horn neurons to cutaneous thermal stimulation and graded electrical stimulation of afferent nerves as well as to excitatory amino acids and also elucidated whether the actions of Ca2+ and Mg2+ are additive or antagonistic. Mg2+ suppressed the thermal and C-fiber responses of wide dynamic range (WDR) cell without any effect on the A-fiber responses. When Mg2+ was directly applied onto the spinal cord, its inhibitory effect was dependent on the concentration of Mg2+ and duration of application. The NMDA- and kainate-induced responses of WDR cell were suppressed by Mg2+, the NMDA-induced responses being inhibited more strongly. Ca2+ also inhibited the NMDA-induced responses current-dependently. Both inhibitory actions of Mg2+ and Ca2+ were additive, while Mg2+ suppressed the EGTA-induced augmentation of WDR cell responses to NMDA and C-fiber stimulation. Magnesium had dual effects on the spontaneous activities of WDR cell. These experimental findings suggest that Mg2+ is implicated in the modulation of pain in the rat spinal cord by inhibiting the responses of WDR cell to noxious stimuli more strongly than innocuous stimuli.

Calcium modulates excitatory amino acid (EAA)- and substance P-induced rat dorsal horn cell responses

Excitatory amino acid (EAA) and substance P (SP) have been known to be primary candidates for nociceptive neurotransmitter in the spinal cord, and calcium ions are implicated in processing of the sensory informations mediated by EAA and SP in the spinal cord. In this study, we examined how Ca2+ modified the responses of dorsal horn neurons to single or combined iontophoretical application of EAA and SP in the rat. All the LT cells tested responded to kainate, whereas about 55% of low threshold (LT) cells responded to iontophoretically applied NMDA. NMDA and kainate excited almost all wide dynamic range (WDR) cells. These NMDA- and kainate-induced WDR cell responses were augmented by iontophoretically applied EGTA, but suppressed by Ca2+, Mn2+ verapamil and omega-conotoxin GVTA, effect of verapamil being more prominent and well sustained. Ca2+ and Mn2+ antagonized the augmenting effect of EGTA. On the other hand, prolonged spinal application of EGTA suppressed the response of WDR cell to NMDA. SP had triple effects on the spontaneous activity as well as NMDA-induced responses of WDR cells: excitation, inhibition and no change. EGTA augmented, but Ca2+, Mn2+ and verapamil suppressed the increase in the NMDA-induced responses and spontaneous activities of WDR cells following iontophoretical application of SP. These results suggest that in the spinal cord, sensory informations mediated by single or combined action of EAA and SP can be modified by the change in calcium ion concentration.

Dopaminergic inhibition of dorsal horn cell activity in the cat

Dopamine has been generally known to exert antinociceptive action in behavioral pain test, such as tail flick and hot plate test, but there appears to be a great variance in the reports on the antinociceptive effect of dopamine depending on the dosage and route of drug administration and type of animal preparation. In the present study, the effects of dopamine on the responses of wide dynamic range (WDR) cells to mechanical, thermal and graded electrical stimuli were investigated, and the dopamine-induced changes in WDR cell responses were compared between animals with an intact spinal cord and the spinal animals. Spinal application of dopamine (1.3 & 2.6 mM) produced a dose-dependent inhibiton of WDR cell responses to afferent inputs, the pinch-induced or the C-fiber evoked responses being more strongly depressed than the brush-induced or the A-fiber evoked responses. The dopamine-induced inhibition was more pronounced in the spinal cat than in the cat with intact spinal cord. The responses of WDR cell to thermal stimulation were also strongly inhibited. Dopamine D2 receptor antagonist, sulpiride, but not D1 receptor antagonist, significantly blocked the inhibitory action of dopamine on the C-fiber and thermal responses of dorsal horn cells. These findings suggest that dopamine strongly suppresses the responses of WDR cells to afferent signals mainly through spinal dopamine D2 receptors and that spinal dopaminergic processes are under the tonic inhibitory action of the descending supraspinal pathways.

Calcium channel blockers suppress the responses of rat dorsal horn cell to nociceptive input

Calcium ions are implicated in a variety of physiological functions, including enzyme activity, membrane excitability, neurotransmitter release, and synaptic transmission, etc. Calcium antagonists have been known to be effective for the treatment of exertional angina and essential hypertension. Selective and nonselective voltage-dependent calcium channel blockers also have inhibitory action on the acute and tonic pain behaviors resulting from thermal stimulation, subcutaneous formalin injection and nerve injury. This study was undertaken to investigate the effects of iontophoretically applied Ca++ and its antagonists on the responses of WDR (wide dynamic range) cells to sensory inputs. The responses of WDR cells to graded electrical stimulation of the afferent nerve and also to thermal stimulation of the receptive field were recorded before and after iontophoretical application of Ca++, EGTA, Mn++, verapamil, omega-conotoxin GVIA, omega-conotoxin MVIIC and omega-agatoxin IVA. Also studied were the effects of a few calcium antagonists on the C-fiber responses of WDR cells sensitized by subcutaneous injection of mustard oil (10%). Calcium ions and calcium channel antagonists (Mn++, verapamil, omega-conotoxin GVIA & omega-agatoxin IVA) current-dependently suppressed the C-fiber responses of WDR cells without any significant effects on the A-fiber responses. But omega-conotoxin MVIIC did not have any inhibitory actions on the responses of WDR cell to A-fiber, C-fiber and thermal stimulation. Iontophoretically applied EGTA augmented the WDR cell responses to C-fiber and thermal stimulations while spinal application of EGTA for about 20 ~ 30 min strongly inhibited the C-fiber responses. The augmenting and the inhibitory actions of EGTA were blocked by calcium ions. The WDR cell responses to thermal stimulation of the receptive field were reduced by imtophoretical application of Ca++, verapamil, omega -agatoxin IVA, and omega-conotoxin GVIA but not by omega-conotoxin MVIIC. The responses of WDR cells to C-fiber stimulation were augmented after subcutaneous injection of mustard oil (10%, 0.15 ml) into the receptive field and these sensitized C-fiber responses were strongly suppressed by iontophoretically applied Ca++, verapamil, omega-conotoxin GVIA and omega-agatoxin IVA. These experimental findings suggest that in the rat spinal cord, L-, N-, and P-type, but not Q-type, voltage-sensitive calcium channels are implicated in the calcium antagonist-induced inhibition of the normal and the sensitized responses of WDR cells to C-fiber and thermal stimulation, and that the suppressive effect of calcium and augmenting action of EGTA on WDR cell responses are due to changes in excitability of the cell.