The cellular and molecular mechanisms of bone invasion by oral squamous cell carcinoma.

Oral squamous cell carcinomas (SCCs) are malignant tumours that frequently invade the mandibular bone and bone invasion is a common clinical problem. Recent studies have revealed that bone resorption by osteoclasts is an important step in the process of bone invasion by oral SCCs. However, the cellular and molecular mechanisms of bone invasion by oral SCCs remain unclear. Oral SCCs invade the mandibular bone through an erosive, mixed or infiltrative pattern that correlates with clinical behaviours. The expressions of interleukin (IL)-6, IL-11, tumour necrosis factor (TNF) α and parathyroid hormone-related protein (PTHrP) were higher in the infiltrative pattern than in the erosive pattern. These cytokines lead to receptor activator of NF-κB ligand (RANKL) expression or osteoprotegerin (OPG) suppression not only in oral SCC cells but also in cancer stromal cells to induce osteoclastogenesis. Taken together, oral SCCs provide a suitable microenvironment for osteoclastogenesis to regulate the balance of RANKL and OPG. In this review, we introduce recent advances in the knowledge of the cellular and molecular mechanisms, by which oral SCC invades mandibular bone based on the recent findings of our lab and others.

Mitogenesis in response to PDGF and bombesin abolished by microinjection of antibody to PIP2.

The turnover of phosphatidylinositol 4,5-bisphosphate (PIP2) is believed to constitute a crucial step in the signaling pathways for stimulation of cells by a variety of bioactive substances, including mitogens, but decisive evidence for the idea has not been obtained. In the present study, a monoclonal antibody to PIP2 was microinjected into the cytoplasm of NIH 3T3 cells before or after exposure to mitogens. The antibody completely abolished nuclear labeling with [3H]thymidine induced by platelet-derived growth factor and bombesin, but not by fibroblast growth factor, epidermal growth factor, insulin, or serum. The findings strongly suggest that PIP2 breakdown is crucial in the elicitation and sustaining of cell proliferation induced by some types of mitogens such as platelet-derived growth factor and bombesin.

Galanin inhibits neural activity in the subfornical organ in rat slice preparation.

The activation of the subfornical organ (SFO), a circumventricular organ, induces water intake and vasopressin release. Since central administrations of galanin (GAL) suppress water intake and vasopressin release, GAL may inhibit the neural activity of SFO neurons. In the present study, we investigated effects of GAL on the SFO using molecular biological, electrophysiological and anatomical techniques. Reverse transcription-polymerase chain reaction analysis demonstrated the presence in the SFO of rats of the mRNAs for each of the three known GAL receptor subtypes (GalR1, GalR2 and GalR3). In extracellular recordings in SFO slice preparations, GAL dose-dependently inhibited the neural activity of cells from a number of recording sites. Many GAL-sensitive SFO neurons showed excitatory responses to angiotensin II (ANGII). The GalR1 agonist M617 inhibited the activity of SFO neurons, whereas the GalR2 and GalR3 agonist GAL(2-11) had almost no effect. In patch-clamp recordings, GAL induced an outward current in SFO neurons without influencing synaptic currents. An immunoelectron microscopic study revealed the existence of GAL-containing synaptic vesicles in the SFO. These results suggest that the SFO has neural inputs involving GAL. The response to GAL is inhibitory, mediated at least in part by GalR1 and provides a plausible explanation for the opposite effects of ANGII and GAL seen in vivo on water intake and vasopressin release.

MS-275, a histone deacetylase inhibitor, selectively induces transforming growth factor beta type II receptor expression in human breast cancer cells.

Transcriptional repression of the transforming growth factor (TGF)-1P type II receptor (TPRII) gene appears to be a major mechanism to inactivate TGF-beta responsiveness in many human cancers. Because histone acetylation/deacetylation plays a role in transcriptional regulation, we have examined the effect of MS-275, a synthetic inhibitor of histone deacetylase, in human breast cancer cell lines. MS-275 showed antiproliferative activity against all human breast cancer cell lines examined and induced TbetaRII mRNA, but not TGF-beta type I receptor mRNA. MS-275 caused an accumulation of acetylated histones H3 and H4 in total cellular chromatin. An increase in the accumulation of acetylated histones H3 and H4 was detected in the TbetaRII promoter after treatment with MS-275. However, the level of histone acetylation did not change in chromatin associated with the TGF-beta type I receptor gene. MS-275 treatment enhanced TGF-beta1-induced plasminogen activator inhibitor 1 expression. Thus, antitumor activity of MS-275 may be mediated in part through the induction of TbetaRII expression and consequent potentiation of TGF-beta signaling.

Potent antitumor activity of MS-247, a novel DNA minor groove binder, evaluated by an in vitro and in vivo human cancer cell line panel.

We synthesized a novel anticancer agent MS-247 (2-[[N-[1-methyl-2-[5-[N-[4-[N,N-bis(2-chloroethyl) amino] phenyl]] carbamoyl]-1H-benzimidazol-2-yl] pyrrol-4-yl] carbamoyl] ethyldimethylsulfonium di-p-toluenesulfonate) that has a netropsin-like moiety and an alkylating residue in the structure. We evaluated antitumor activity of MS-247 using a human cancer cell line panel coupled with a drug sensitivity database and subsequently using human cancer xenografts. The average MS-247 concentration required for 50% growth inhibition against a panel of 39 cell lines was 0.71 microM. The COMPARE analysis revealed that the differential growth inhibition pattern of MS-247 significantly correlated with those of camptothecin analogues and anthracyclins, indicating that MS-247 and the two drug groups might have similar modes of action. MS-247 exhibited remarkable antitumor activity against various xenografts. A single i.v. injection of MS-247 significantly inhibited the growth of all 17 xenografts tested, which included lung, colon, stomach, breast, and ovarian cancers. In many cases, MS-247 was more efficacious than cisplatin, Adriamycin, 5-fluorouracil, cyclophosphamide, VP-16, and vincristine and was almost comparable with paclitaxel and CPT-11; these are the most clinically promising drugs at present. MS-247 was noticeably more effective than paclitaxel (in HCT-15) and CPT-11 (in A549, HBC-4, and SK-OV-3). The toxicity of MS-247, indicated by body weight loss, was reversible within 10 days after administration. The MS-247 mode of action showed DNA binding activity at the site where Hoechst 33342 bound, inhibited topoisomerases I and II (as expected by the COMPARE analysis) blocked the cell cycle at the G2-M phase, and induced apoptosis. These results indicate that MS-247 is a promising new anticancer drug candidate to be developed further toward clinical trials.

Molecular cloning and nucleotide sequences of cDNAs specific for rat liver ribosomal proteins S17 and L30.

cDNA clones coding for rat liver ribosomal proteins S17 and L30 have been isolated by positive hybridization-translation assay from a cDNA library prepared from 8-9S poly(A)+RNA from free polysomes of regenerating rat liver. The cDNA clone specific for S17 protein (pRS17-2) has a 466-bp insert with the poly(A) tail. The complete amino acid (aa) sequence of S17 protein was deduced from the nucleotide sequence of the cDNA. S17 protein consists of 134 aa residues with an Mr of 15 377. The N-terminal aa sequence of S17 protein determined by automatic Edman degradation is consistent with the sequence data. The aa sequence of S17 shows strong homology (76.9%) to that of yeast ribosomal protein 51 [Teem and Rosbash, Proc. Natl. Acad. Sci. USA 80 (1983) 4403-4407] in the two-thirds N-terminal region. The cDNA clone specific for L30 protein (pRL30) has a 394-bp insert. The aa sequence of L30 protein was deduced from the nucleotide sequence of the cDNA. The protein consists of 114 aa residues with an Mr of 12 652. When compared with the N-terminal aa sequence of rat liver L30 protein [Wool, Annu. Rev. Biochem. 48 (1979) 719-754], pRL30 was found not to contain the initiation codon and 5'-noncoding region. The cDNA showed twelve silent changes in the coding region, one point mutation and one base deletion in the 3'-noncoding region, compared with mouse genomic DNA for L30 protein [Wiedemann and Perry, Mol. Cell Biol. 4 (1984) 2518-2528].

Synthesis and histone deacetylase inhibitory activity of new benzamide derivatives.

Newly synthesized benzamide derivatives were evaluated for their inhibitory activity against histone deacetylase. The structure of these derivatives was unrelated to the known inhibitors, and IC(50) values of the active compounds were in the range of 2-50 microM. Structure-activity relationship on the benzanilide moiety showed that the 2'-substituent, an amino or hydroxy group, was indispensable for inhibitory activity. Although the electronic influence of the substituent in the anilide moiety showed only a small effect on inhibitory activity, the steric factor in the anilide moiety, especially at positions 3'and 4', played an important role in interaction with the enzyme. Among these benzamide derivatives, MS-275 (1), which showed significant antitumor activity in vivo, has been selected for further investigation.

Molecular cloning and nucleotide sequence of DNA complementary to rat ribosomal protein S26 messenger RNA.

A cDNA clone specific for rat ribosomal protein S26 was isolated by a positive hybridization translation assay from a cDNA library made for 8-9S poly(A)mRNA from regenerating rat liver. The nucleotide sequence of the cDNA was determined. The sequence contains 23 base pairs in the 5' noncoding region, 345 base pairs in the protein coding region and 67 base pairs in the 3' noncoding region besides the poly(A) tail. The primary structure of the protein S26 was deduced from the nucleotide sequence. It consists of 115 amino acids. Its molecular weight is 13,015 and its pI is about 11.1. The calculated amino acid composition is consistent with the reported composition of S26. From the results of Southern blot analysis, the protein S26 appears to have multiple genes.

A novel synthetic DNA minor groove binder, MS-247: antitumor activity and cytotoxic mechanism.

PURPOSE: MS-247 is a novel synthetic compound possessing a DNA-binding moiety and a DNA-alkylating residue, chlorambucil. In this study, we evaluated the antitumor activity of MS-247 against murine tumor cell lines and its effects on DNA molecules in both cell-free and cellular systems. METHODS: The in vitro cytotoxic activity of MS-247 was evaluated against four murine tumor cell lines, P388, L1210, Colon26 and B16, and its in vivo antitumor activity was also tested in comparison with Adriamycin (ADM), cisplatin (CDDP) and paclitaxel. The ability of MS-247 to associate with the DNA minor groove was assessed by measuring quenching of Hoechst 33342 fluorescence. DNA-DNA interstrand crosslinks (ICL) were detected by an alkaline elution assay for cellular DNA and a band-shift assay using the plasmid pBR322. The effects of MS-247 on macromolecule synthesis (DNA, RNA and proteins) were examined by measuring incorporation of the radiolabeled precursors. RESULTS: MS-247 exhibited in vitro cytotoxicity with IC(50) values ranging 11 to 500 nM, and MS-247 given i.v. showed strong in vivo antitumor activity against i.p.-implanted L1210 leukemia cells and s.c.-implanted Colon26 carcinoma cells, and moderate activity against i.p.-implanted P388 leukemia cells but no apparent activity against s.c.-implanted B16 melanoma cells. MS-247 reversibly displaced Hoechst 33342 bound to DNA within a few minutes, and irreversibly formed ICL within 1-6 h in both the cell-free system and the cellular system. These results suggest that an association of MS-247 with the DNA minor groove occurred more quickly than ICL formation. The inhibition of DNA synthesis was more prominent than the inhibition of RNA and protein synthesis in L1210 cells exposed to MS-247, and a 6-h incubation with MS-247, which formed apparent ICL in the cellular system, strongly inhibited DNA synthesis. This result suggests that impairment of DNA replication preceded the inhibition of RNA and protein synthesis and that ICL formation greatly contributed to the inhibition of macromolecule synthesis. CONCLUSION: The results of this study suggest that MS-247 exerts its cytotoxic effect through impairment of DNA function by getting into the minor groove of DNA and subsequently forming ICL. MS-247 has potent antitumor activity with a different spectrum from the activity of clinically proven antitumor agents such as paclitaxel, ADM and CDDP against several murine tumor cell lines. This result suggests that MS-247 may be useful for the treatment of human cancers.

Reversal of multidrug resistance by a novel quinoline derivative, MS-209.

MS-209, a novel quinoline derivative, was examined for its reversing effect on multidrug-resistant tumor cells. MS-209 at 1-10 microM completely reversed resistance against vincristine (VCR) in vitro in multidrug-resistant variants of mouse leukemia P388 cells (VCR-resistant P388/VCR and Adriamycin (ADM)-resistant P388/ADM) and human leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM). MS-209 at 1-10 microM also completely reversed resistance against ADM in vitro in P388/VCR cells, K562/VCR cells, and K562/ADM cells. In ADM-resistant P388 (P388/ADM) cells, however, ADM resistance was only partially reversed at the MS-209 concentrations tested. MS-209 enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing mice. When MS-209 was given p.o. at 80 mg/kg twice a day (total dose, 160 mg/kg per day) with 100 micrograms/kg VCR, a treated/control (T/C) value of 155% was obtained. MS-209 also enhanced the chemotherapeutic effect of ADM in P388/ADM-bearing mice. The most prominent effects were obtained when MS-209 was given with 2 mg/kg ADM, yielding T/C values of 150%-194% for the combined treatment at an MS-209 dose of 200-450 mg/kg. MS-209 inhibited [3H]-azidopine photolabeling of P-glycoprotein efficiently. Furthermore, the accumulation of ADM in K562/ADM cells was increased more efficiently by MS-209 than by verapamil. These results indicate that MS-209, like verapamil, directly interacts with P-glycoprotein and inhibits the active efflux of antitumor agents, thus overcoming multidrug resistance in vitro and in vivo.

Relationship between multidrug resistant gene expression and multidrug resistant-reversing effect of MS-209 in various tumor cells.

MS-209 is a novel quinoline compound which can overcome multidrug resistance (MDR) both in vitro and in vivo, while having a low level of side effects, and is now being evaluated in a clinical phase II study. Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantitate the expression levels of MDR genes in various mouse and human tumor cell lines. The MDR gene and the beta actin gene, as the internal reference standard, were coamplified separately, and the relative expression of the MDR gene was represented by the MDR/beta actin ratio. The in vitro MDR-reversing effect of MS-209 was then compared with the MDR gene expression (MDR/beta actin ratio). We found a significant correlation between these two parameters. Moreover, a significant correlation was also observed between the level of expression of the MDR1 gene and that of P-glycoprotein in human cell lines. Therefore, the efficacy of MS-209 seems to specifically depend on the level of MDR gene expression (P-glycoprotein). From these observations, it is suggested that RT-PCR assays of MDR1 gene in tumor biopsy specimens might be an effective means to predict the response of tumor cells to combination therapy with MS-209.

Potentiation of the antitumor activity by a novel quinoline compound, MS-209, in multidrug-resistant solid tumor cell lines.

A novel quinoline compound, MS-209, was examined for its ability to reverse multidrug resistance (MDR) in several murine and human MDR solid tumor cell lines both in vitro and in vivo. MS-209 strongly reversed drug resistance to adriamycin (ADM) and vincristine (VCR) in acquired MDR tumor cell lines, 2780AD and KB-C1. In addition, MS-209 enhanced the cytotoxic effect of ADM and VCR on various human and murine cell lines. Particularly in 4-1St cells, which are extremely resistant to ADM and VCR, MS-209 at a concentration of 3 microM enhanced the cytotoxicity of ADM and VCR, 88- and 350-fold, respectively. MS-209 administered orally, together with ADM, enhanced the antitumor activity of ADM on Colon 26 and 4-1St tumors implanted subcutaneously (SC) in mice; the antitumor effect of ADM plus MS-209 was higher than that of ADM alone at the maximum tolerated dose (MTD). Furthermore, the coadministration schedules of MS-209 to attain the highest potentiation of ADM activity were examined using Colon 26 tumors. The maximum antitumor activity was obtained when MS-209 was administered on the same day as ADM. MS-209 administered a day before the ADM injection exhibited no potentiation effect, whereas MS-209 administered a day after the ADM injection showed a moderate effect. The effect of MS-209 was weaker when administered in a fractionated manner than when administered as a single dose. The results presented in this article suggest that MS-209 is an effective agent to overcome MDR in cancer chemotherapy.

Development of an orofacial model of acute inflammation in the rat.

An appropriate model was created by the paraperiosteal injection of mustard oil (20% allyl isothiocyanate dissolved in mineral oil) into the periarticular temporomandibular tissue of anaesthetized rats. Inflammation was assessed by the plasma extravasation of Evans' blue dye bound to plasma protein. This was confirmed visually and compared spectrophotometrically with the contralateral untreated control site (p less than 0.0005). A time-course study of the effect of mustard oil on Evans' blue extravasation revealed a gradually increasing effect that was maximal at 30 min after administration, with no further increase at 60 min. A dose-response study showed that giving 30 microliters of 20% mustard oil produced the maximal effect, with no further increase from 50 microliters. To confirm induction of inflammation, polymorphonuclear neutrophil infiltration was assessed morphometrically and found to increase in the treated tissue compared with the contralateral untreated control (p less than 0.001).

Solving the binding problem of the brain with bi-directional functional connectivity.

We propose a neural network model which gives one solution to the binding problem on the basis of 'functional connectivity' and bidirectional connections. Here, 'functional connectivity' is dynamic neuronal connectivity peculiar to temporal spike coding neural networks with coincidence detector neurons. The model consists of a single primary map and two higher modules which extract two different features shown on the primary map. There exist three layers in each higher module and the layers are connected bi-directionally. An object in the outer world is represented by a 'global dynamical cell assembly' which is organized across the primary map and the two higher modules. Detailed, but spatially localized, information is coded in the primary map, whereas coarse, but spatially extracted information or globally integrated information is coded in the higher modules. Computer simulations of the proposed model show that multiple cell assemblies sharing the same neurons partially can co-exist. Furthermore, we introduce a three-dimensional J-PSTH (Joint-Peri Stimulus Time Histogram) which is capable of tracking such cell assemblies, altering its constituent neurons as in our proposed model.

Effects of midazolam on pain sensations in the face.

OBJECTIVES: This study investigated the effects of midazolam, a sedative, on tactile and pain sensations on the skin of the chin. STUDY DESIGN: Thirty-seven volunteers were segregated into four groups; the first group was the control group; the second to fourth groups were administered 0.025 mg/kg, 0.05 mg/kg, and 0.075 mg/kg of midazolam, respectively, as a bolus injection. All volunteers reclined in a dental chair for the experiment. Tactile and pain sensations were determined over time after injection of midazolam, the former using von Fray thread, the latter using an esthesiometer. RESULTS: Thresholds of tactile sensitivity and of pain were statistically significantly different from control values at 10 minutes after injection of midazolam in the 0.05 mg/kg group and in the 0.075 mg/kg group. CONCLUSION: Although 0.025 mg/kg of midazolam produced sedation, at least 0.05 mg/kg of this agent was required to alter the thresholds for perception of tactile and painful stimulation.

Increase in the threshold of pain and touch sensation in the human face with clonidine plus 30% nitrous oxide.

OBJECTIVE: This study was undertaken to assess the effects of clonidine combined with 30% nitrous oxide on tactile and pain sensations in the human face. STUDY DESIGN: Thirty-three subjects were involved in the study. The subjects were divided into 4 groups: 100% oxygen with placebo; 30% N2O with placebo; 100% oxygen with clonidine (0.075 mg), and 30% N2O with clonidine. Three tests for the threshold of pain sensation and tactile sensation were made at 60 minutes before and 0, 15, and 30 minutes during N2O or O2 inhalation. RESULTS: (1) The N2O with clonidine significantly increased the threshold of pain and tactile sensation in comparison with the other 3 treatments. (2) In terms of pain sensation, both N2O and clonidine showed significant increases in threshold of pain in comparison with the control values. CONCLUSIONS: These results indicate that the analgesic effects of 30% nitrous oxide are enhanced when use of the gas is combined with prior clonidine administration.

The effects of idazoxan combined with 30% nitrous oxide on the jaw-opening reflex in the rat.

In rats, the jaw-opening reflex is elicited by activation of a nociceptive receptor by the electric stimulation of the tooth pulp. This study was undertaken to assess the effects of 30% nitrous oxide and 30% nitrous oxide with idazoxan, an alpha 2-adrenergic antagonist, on this reflex. Each rat received electric stimulation for the jaw-opening reflex at 3, 5, 7, 10, 15, and 20 min after both the start of inhalation and the withdrawal of 100% oxygen or 30% nitrous oxide in oxygen. Idazoxan, 400 micrograms/ kg, was administered intravenously at the start of the inhalation period. Amplitudes significantly decreased during inhalation of nitrous oxide, but they returned gradually to control levels after cessation of nitrous oxide inhalation. In the cases of 100% oxygen, 100% oxygen with idazoxan, and 30% nitrous oxide in oxygen with idazoxan, amplitudes did not change from controls during and after 30% nitrous oxide inhalation. The latency remained unchanged irrespective of the treatment. Since in rats the degree of inhibition by 30% nitrous oxide in oxygen is partially diminished by administration of idazoxan, we conclude that nitrous oxide affects an alpha 2-adrenergic receptor in the central nervous system.

Efficacy of mandibular topical anesthesia varies with the site of administration.

This study compared the threshold of pain sensitivity in the anterior mandibular mucobuccal fold with the posterior. This was followed by a comparison of the reduction of needle insertion pain in the anterior mucobuccal fold and the pterygo-temporal depression by either topical anesthesia or nitrous oxide inhalation. The pain threshold was determined by an analgometer, a pain-measuring device that depends on pressure readings; additionally, pain caused by a needle inserted by a normal technique was assessed using a visual analog scale (VAS). The threshold of pain was significantly lower in the incisor and canine regions than in the premolar and the molar regions (P < 0.001). Compared to a placebo, topical anesthesia significantly reduced the pain from needle insertion in the mucobuccal fold adjacent to the mandibular canine (P < 0.001), but did not significantly reduce pain in the pterygotemporal depression. The addition of 30% nitrous oxide did not significantly alter pain reduction compared to a control of 100% oxygen. These results suggest that topical anesthesia application may be effective in reducing the pain of needle insertion in the anterior mandibular mucobuccal fold, but may not be as effective for a standard inferior alveolar nerve block. The addition of 30% nitrous oxide did not lead to a significant improvement.

[Effects of isosorbide dinitrate on regional cerebral blood flow and intracranial pressure in cats].

The effects of isosorbide dinitrate (ISDN) on regional cerebral blood flow (rCBF) and intracranial pressure (ICP) were examined in cats. A low dose of ISDN (2.5 micrograms/kg/min) infusion did not show any changes in cerebral hemodynamics. During high dose of ISDN (5.0 micrograms/kg/min) or NTP (5.0 micrograms/kg/min) infusion, mean blood pressure (mBP) decreased by 10 to 20% accompanied by decreased cerebral perfusion pressure (CPP: mBP-ICP), however, rCBF or ICP did not change. It is concluded that intravenous administrations of ISDN in a dose of 2.5-5.0 micrograms/kg/min that produce slight decrease in blood pressure did not influence on cerebral hemodynamics.

[Differential effects of isosorbide dinitrate and nitroprusside on pial vessel diameter in cats].

Changes in pial vessel diameter combined with regional cerebral blood flow (CoBF) during infusion of vasodilating drugs, isosorbide dinitrate (ISDN) 5 micrograms/kg/min and nitroprusside (NTP) 5 micrograms/kg/min, compared with haemorrhagic hypotension were studied in cats anesthetized with halothane (1.0%). Pial arteries and veins were measured by image-splitting technique and were each divided into three groups according to the reference diameter: I; < 50 microns, II; 51 < microns < 100, III; 101 < microns. With either drug, the mean blood pressure (mBP) decreased by 10-20%. There was significant decrease in cerebral vascular resistance with ISDN compared with haemorrhagic hypotension while CoBF (H2 clearance) remained unchanged. Dilatation of pial arteries depending on vessel size with ISDN was two-hold compared with haemorrhagic hypotension without any change in all veins. Consistent and significant dilation of veins (I and II) was observed only during NTP infusion. These findings indicate the differential effect of ISDN and NTP on pial arteries and veins.