A role for dorsal and ventral hippocampus in response learning.

The hippocampus and the striatum have been traditionally considered as part of different and independent memory systems despite growing evidence supporting that both brain regions may even compete for behavioral control in particular learning tasks. In this regard, it has been reported that the hippocampus could be necessary for the use of idiothetic cues in several types of spatial learning tasks. Accordingly, the ventral striatum receives strong anatomical projections from the hippocampus, suggesting a participation of both regions in goal-directed behavior. Our work examined the role of the dorsal and ventral hippocampus on a response learning task. Cytochrome c oxidase (C.O.) quantitative histochemistry was used as an index of brain oxidative metabolism. In addition, determination of C.O. subunit I levels in the hippocampus by western blot analysis was performed to assess the contribution of this subunit to overall C.O. activity. Increased brain oxidative metabolism was found in most of the studied hippocampal subregions when experimental group was compared with a swim control group. However, no differences were found in the amount of C.O. subunit I expressed in the hippocampus by western blot analysis. Our results support that both the dorsal and ventral hippocampus are associated with the use of response strategies during response learning.

Polyinosinic acid induces TNF and NO production as well as NF-kappaB and AP-1 transcriptional activation in the monocytemacrophage cell line RAW 264.7.

OBJECTIVE: This study evaluates the poly inosinic acid (poly I)-induced activation in the murine monocytemacrophage cell line RAW 264.7, which led to an inflammatory phenotype. MATERIAL: RAW 264.7, and WEHI 164 cell lines were used. RESULTS: The activation process is characterized by the acquisition of a mature macrophage morphology and the production of inflammatory mediators tumor necrosis factor (TNF) and nitric oxide (NO). The activation by poly I has distinctive features. Thus, poly I induced an increase in nuclear factor kappaB (NF-kappaB) transcriptional activity due to a long-term degradation of inhibitory NF-kappaB (IkappaB) beta while lipopolysaccharide (LPS) induced the degradation of both IkappaBalpha and IkappaBbeta. Poly I also induced an increase in activator protein 1 (AP-1) transcriptional activity, possibly due to the activation of the mitogen activated protein kinases (MAPKs) ERK, Jun N terminal kinase (JNK) and p38. Dextran sulphate (DS) efficiently inhibited the activation induced by poly I including the production of the inflammatory mediators. Dextran sulphate also inhibited AP-1 and NF-kappaB transcriptional activities in poly I-stimulated cells. RAW 264.7 cells express macrophage scavenger receptor 1 (Msr1) type I and Msr1 type II that are differently up-regulated upon treatment with poly I. CONCLUSIONS: The results presented demonstrate that the well-known blocker of scavenger receptors poly I activates macrophages to produce TNF and NO, triggering specific signal transduction pathways.

Variability of genetic alterations in different sites of head and neck cancer.

OBJECTIVE: Tumors arising from different sites of the head and neck area have different clinical behavior. However, most of the studies on genetic alterations in head and neck squamous cell carcinomas do not make a distinction between the sites within this area. The objective of this study is to compare the genetic alterations in three different sites of the head and neck (larynx, oropharynx, and hypopharynx). STUDY DESIGN: Prospective study. METHODS: Thirty-eight laryngeal, 29 oropharyngeal, and 37 hypopharyngeal carcinomas were studied. DNA from tumor and healthy tissue was evaluated for amplification of the oncogenes at 11q13 region (CCND1, FGF3, FGF4 and EMS1) and of the oncogenes MYC and ERBB1; for integration of the human papillomavirus (HPV) types 6b and 16; for loss of heterozygosity (LOH) at p53 and NAT2; and for the cellular DNA content. RESULTS: FGF3 and FGF4 showed a significantly higher frequency of amplification in hypopharyngeal tumors (P =.006 and P =.0002, respectively). CCND1 amplification had a nearly statistically significant (P =.072) higher frequency of amplification in hypopharyngeal tumors. Aneuploid tumors were found in a significantly lower proportion in the larynx (P =.03) compared with the other sites. For the other genetic alterations, no significant differences among the three sites were found. CONCLUSIONS: These results suggest that cancers originating from different sites in the head and neck may have different tumor biology. Therefore, they should be considered as different entities.

EMS1 gene amplification correlates with poor prognosis in squamous cell carcinomas of the head and neck.

The relationship between CCND1 and/or EMS1 amplification and disease outcome was studied in a prospective series of 104 head and neck squamous cell carcinomas treated by surgical resection. The CCND1 and EMS1 copy number in tumor samples was estimated by differential PCR. The presence or absence of amplification was analyzed in relation to clinicopathological variables, tumor recurrence, and patient survival. CCND1 amplification occurred in 32 cases (31%) and was associated with increased lymph node stage (P = 0.005) and advanced disease stage (P = 0.003). EMS1 amplification was identified in 21 cases (20%) and was related with advanced T stages (P = 0.001), increased lymph node stage (P = 0.02), advanced disease stage (P = 0.041), poor histological differentiation (P = 0.018), recurrent disease (P = 0.0004), and reduced disease-specific survival (P < 0.0001). Coamplification of both genes occurred in 11 cases (11.5%). Multivariate analysis confirmed that in addition to regional lymph node status, EMS1 amplification is an independent predictor of death from the tumor (P = 0.0027). CCND1 amplification was not prognostic. These data indicate that EMS1 amplification, but not CCND1 amplification, predicts early recurrence and reduced survival in squamous cell carcinoma of the head and neck. The prognostic significance previously attributed to CCND1 amplification may be attributable to its frequent coamplification with EMS1.

Relationship of human papillomavirus to ploidy in squamous cell carcinomas of the head and neck.

To establish the relationship between the presence of human papillomavirus (HPV) gene sequences and the development of genetic abnormalities, 31 squamous cell carcinomas of the head and neck were studied for the presence of HPV types 6b and 16 and the DNA content by flow cytometry. Eighteen (58%) cases were aneuploid. HPV DNA was present in seven (22.5%) tumors. Five of them were positive for the HPV type 6b and two for the HPV type 16. Aneuploidy was correlated with poorly differentiated tumors. No correlation was found between the presence of HPV, DNA content, or tumor differentiation. Consequently, the presence of HPV gene sequences does not seem to be related to a higher incidence of genetic abnormalities in squamous cell carcinomas of the head and neck.

Melatonin blocks the activation of estrogen receptor for DNA binding.

The present study shows that melatonin prevents, within the first cell cycle, the estradiol-induced growth of synchronized MCF7 breast cancer cells. By using nuclear extracts of these cells, we first examined the binding of estradiol-estrogen receptor complexes to estrogen-responsive elements and found that the addition of estradiol to whole cells activates the binding of the estrogen receptor to DNA whereas melatonin blocks this interaction. By contrast, melatonin neither affects the binding of estradiol to its receptor nor the receptor nuclear localization. Moreover, we also show that addition of estradiol to nuclear extracts stimulates the binding of estrogen receptor to DNA, but this activation is also prevented by melatonin. The inhibitory effect caused by melatonin is saturable at nanomolar concentrations and does not appear to be mediated by RZR nuclear receptors. The effect is also specific, since indol derivatives do not cause significant inhibition. Furthermore, we provide evidence that melatonin does not interact with the estrogen receptor in the absence of estradiol. Together, these results demonstrate that melatonin interferes with the activation of estrogen receptor by estradiol. The effect of melatonin suggests the presence of a receptor that, upon melatonin addition, destabilizes the binding of the estradiol-estrogen receptor complex to the estrogen responsive element.

The mouse tumor necrosis factor receptor 2 gene: genomic structure and characterization of the two transcripts.

The mouse TNFR2 gene has been cloned, sequenced, and characterized as a gene spanning >44 kb of the genome. By alignment of five genomic clones we have established that TNFR2 consists of 10 exons and 9 introns with exons ranging in size from 35 bp to 2.6 kb and introns ranging from 322 bp to >16 kb. All splice acceptor and donor sites conform to the canonical AG/GT rule. The translation initiation and termination sites are located in exon 1 and 10, respectively. Although TNFR2 lacks a canonical TATA box, the gene is transcribed from a unique start site located 70 bp upstream of the ATG initiation codon that conforms to the consensus Inr motif. Several cis-elements for transcription factors were identified in the 5' flanking region, including NF-1, Sp-1, AP2, gamma-IRE, and NF-kappaBeta motifs. Functional analysis indicates that the region -705/-412 contains a negative cis-acting element and that the minimal promoter contains motifs that confer LPS inducibility. Two mouse TNFR2 mRNAs of 3.2 and 4.1 kb are detected by Northern blot analysis, but until now their origin has not been explained. No evidence of alternative splicing of the coding exons was found. However, hybridization studies and amplification of cDNA ends suggest the use of a noncanonical polyadenylation signal in the untranslated region of exon 10. A comparative analysis of the 3' untranslated regions of the human and mouse TNFR2 genes shows highly divergent 3' ends. The possibility of an ancestral mouse TNFR2 mRNA similar to the short transcript is discussed.

Amplification of ERBB oncogenes in squamous cell carcinomas of the head and neck.

The activation of ERBB oncogenes has been described in various human tumours, including squamous cell carcinomas of the head and neck (SCCHN), and, in some of them, it has been correlated with a poor prognosis. Tissue samples from 59 patients with SCCHN were studied. After DNA extraction, the ERBB1, ERBB2 and ERBB3 copy number in tumour samples was estimated with the polymerase chain reaction (PCR) method. The PCR products were analysed by agarose gel electrophoresis and quantified by image analysis techniques. 9 (15%) cases presented with ERBB1 amplification, which was correlated with lymph node involvement (P = 0.04), poorly differentiated tumours (P = 0.03) and a hypopharyngeal primary site (P = 0.035). No correlation among amplification status, recurrence, metastases and survival was observed, although this may be due to the small number of patients in the amplified group. None of the 59 cases presented amplification of ERBB2 and ERBB3 oncogenes.

[The relation between human papilloma virus and cellular DNA content in epidermoid carcinoma of the head and neck]. / Relación entre el virus del papiloma humano y el contenido celular de ADN en los carcinomas epidermoides de cabeza y cuello.

The relation between the presence of gene sequences of human papilloma virus (HPV) and the development of abnormalities in cellular DNA content was analyzed in 31 squamous-cell carcinomas of the head and neck. The integration of HPV types 16 and 6b by PCR and DNA content was studied by flow cytometry in 31 specimens from patients with squamous-cell carcinoma of the head and neck. Eighteen (58%) cases were aneuploid. HPV DNA was present in seven tumors (22.5%), five of then HPV-6b and two of them HPV-16. Aneuploidy correlated with poorly differentiated tumors. No correlation was found between HPV integration and either cellular DNA content or the degree of histological tumor differentiation. Therefore, the presence of HPV gene sequences did not seem to be associated with a higher incidence of aneuploidy in squamous-cell carcinoma of the head and neck.

Molecular cloning of a mouse homologue for the Drosophila splicing regulator Tra2.

We report the identification of a mouse cDNA, SIG41, encoding a protein of 288 amino acids that is 45% identical (58% similar) to the Drosophila splicing regulator Tra2. SIG41 cDNA contains four polyadenylation signals whose alternative use gives rise to four types of transcripts (2.1, 2.0, 1.5, and 1.4 kb) in mouse cells. Northern analysis and RT-PCR assays showed that SIG41 mRNA is present in virtually all the cell lines and tissues studied, with remarkable levels of expression in uterus and brain tissues. Differential stability of the SIG41 mRNAs was detected in mouse macrophage cells.

MYC amplification in squamous cell carcinomas of the head and neck.

OBJECTIVES: To establish the frequency of MYC amplification in squamous cell carcinoma (SCC) of the head and neck to evaluate its correlation with clinicopathologic variables that are used in clinical practice. DESIGN: Cohort analytic study. SETTING: University Hospital. PATIENTS: Fifty-nine consecutive patients with SCC of the head and neck. INTERVENTION: Oncologic surgery. MAIN OUTCOME MEASURES: The MYC copy number in tumor samples was estimated with the polymerase chain reaction. The presence or absence of amplification was correlated with the anatomic site, T stage, nodal involvement, pathologic grade, recurrence, distant metastases, and survival. RESULTS: Six SCC specimens (11%) showed MYC amplification. A highly statistical correlation between MYC amplification and T stage was noted (P < .005). Amplification was also significantly correlated with a hypopharyngeal primary site (P < .05). No correlation among amplification status, nodal involvement, pathologic grade, relapse, metastases, and survival was observed. CONCLUSIONS: Amplification of MYC is associated with advanced primary tumors, and it appears to be a late event in the tumorigenesis of SCCS of the head and neck. However, there is no correlation between MYC amplification and prognosis.

Identification of an additional member of the cytochrome c oxidase subunit VIIa family of proteins.

We report the cloning, nucleotide sequence, evolutionary analysis, and intracellular localization of SIG81, a silica-induced cDNA from mouse macrophages. The cDNA encodes a 111-amino acid protein with extensive sequence identity with members of the mammalian cytochrome c oxidase subunit VIIa (COX7a) family. A human SIG81 sequence >80% identical with the mouse cDNA was deducted from homologous sequences in the human expressed tags data base. The deduced aminoterminal region shows features common to mitochondrial targeting sequences. A phylogenetic analysis of the carboxyl-terminal domain homologous to COX7a identifies SIG81 as a divergent member of the family with an ancient origin. Southern blot analysis showed that the mouse genome contains two to three copies of the SIG81 gene. Northern blot analysis revealed that the SIG81 transcript is approximately 1 kb and expressed in every tissue tested, with higher levels of expression observed in kidney and liver. Antibodies raised against a glutathione S-transferase SIG81 fusion protein detected a 13.5-kDa protein that co-fractionates with mitochondrial localized enzymatic activity. Taken together, our data suggest that SIG81 is a novel member of the COX7a family that is constitutively expressed in mouse cells.

Molecular mechanisms of TNFalpha cytotoxicity: activation of NF-kappaB and nuclear translocation.

The murine fibrosarcoma cell line WEHI 164 is well known for its susceptibility to tumor necrosis factor (TNFalpha). We have studied the activation of the transcription factor NF-kappaB when WEHI 164 cells are challenged with TNFalpha. NF-kappaB is retained in the cytoplasm of unchallenged cells by its inhibitor IkappaB-alpha. Upon cellular stimulation, IkappaB-alpha is functionally inactivated and NF-kappaB translocated to the nucleus. The extent of the cytotoxic effect and that of nuclear translocation of NF-kappaB show the same TNFalpha dependence. TNFalpha induces a rapid and transient activation of NF-kappaB in WEHI 164 cells which is followed by a second, long lasting phase in which the amount of NF-kappaB complex in the nucleus remains at about 50% of maximum. Upon TNFalpha treatment, IkappaB-alpha is rapidly degraded. However, newly synthesized IkappaB-alpha can be demonstrated later in the cell cytosol. A persistent nuclear localization of NF-kappaB is an obligatory step for the cytotoxic effect to take place. Thus, WEHI 164 cells treated with TNFalpha for up to 6 h can be rescued as long as NF-kappa relocalizes to the cytoplasm in its inactive form. On the other hand, TNFalpha treatments as short as 15 min cause the cytotoxic effect provided that NF-kappaB remains in the nucleus. The activation of NF-kappaB is controlled by both phosphorylation and proteolysis. The activation of NF-kappaB can be blocked by the cysteine protease inhibitor calpain inhibitor I and the serine protease inhibitor TPCK. Signal-induced phosphorylation of IkappaB-alpha does not lead to the dissociation of the inhibitor from NF-kappaB. Phosphorylation appears to regulate the inhibitory activity of IkappaB-alpha both positively and negatively. since inhibitors of protein kinases have opposite effects. Thus, treatment of cells with staurosporin induced a partial activation of NF-kappaB and was synergistic with TNFalpha induced activation. Calphostin C, on the other hand, can block the activation of NF-kappaB by TNFalpha, also blocking its proteolytic degradation.

Measurement of cytotoxicity by propidium iodide staining of target cell DNA. Application to the quantification of murine TNF-alpha.

A rapid and sensitive method is described for the determination of murine tumor necrosis factor (TNF-alpha), which can be performed in microtiter plates using a fluorescence plate scanner. The method is based on the binding of propidium iodide (PI), a membrane-impermeant dye, to nucleic acids of WEHI 164 cells, whose plasma membrane permeable due to TNF-alpha-induced cell damage. The analytical range for the proposed method is 0.3-200 pg/ml of TNF-alpha after 5 h of incubation. The optimal number of target cells was found to be 4-5X10(4)/well. The variability obtained for the PI assay was 7.6%; lower than that obtained with a commonly employed method in which MTT is used to determine cell viability (11.3%). Thus, the PI assay appears to be a reliable and reproducible method for the determination of biologically active TNF-alpha. The assay can be performed in a few hours and has the advantage over the current MTT and 51Cr-released assays that kinetic studies of TNF-alpha toxicity are possible since it permits multiple, sequenced measurements of cell viability during the incubation of the sample. The method can also be used for the determination of human TNF-alpha.

Differential regulation of the murine ribosomal protein L26 gene in macrophage activation.

In mouse RAW 264.7 macrophages, the gene for ribosomal protein L26 is positively regulated by silica. In order to study L26 gene expression a near full-length cDNA for mouse L26 was isolated and characterized. Sequence analysis revealed that mouse L26 is a 145 amino acid protein highly homologous to other vertebrate L26 proteins. The treatment of RAW 264.7 cells with the inflammatory mediators LPS and IFN gamma induced the expression of L26 mRNA, but the patterns of expression obtained differed markedly from silica. On the contrary, TNF alpha acted as a down-regulator of L26 gene. Our results suggest that the synthesis of ribosomal components in response to macrophage activators is inducer-specific. Mouse genomic DNA analysis revealed the presence of multiple (10-12) sequences related to the L26 gene.

Activation of murine macrophages by silica particles in vitro is a process independent of silica-induced cell death.

We have tested the murine macrophagic cell line RAW 264.7 for its ability to undergo activation after exposure to silica particles in vitro. When exposed to silica under controlled conditions (each cell having access to about 10 silica particles), RAW 264.7 cells were able to phagocytose the particles. Concomitantly, there was a significant increase in tumor necrosis factor alpha (TNF alpha) mRNA accumulation and TNF alpha secretion. The level of TNF alpha production by RAW 264.7 cells increased up to 5-fold 48 h after phagocytosis of silica particles with very low cell toxicity. The phagocytic stimulus did not induce nitric oxide production. When cells were exposed to a higher number of silica particles, cell activation was attained at shorter times but a substantial number of cells were damaged at 48 h. Interferon gamma (IFN gamma) alone induced an increased production of TNF alpha in RAW 264.7 cells, not further augmented by a subsequent exposure to silica of the IFN gamma-treated cells. Other macrophage-like cell lines as well as primary peritoneal macrophages were able to phagocytose silica particles but showed different abilities to produce and secrete TNF alpha once phagocytosis took place. Therefore, RAW 264.7 cells were chosen as a model for in vitro studies of the long-term response of macrophages to silica.

[Analysis of rearrangements in the first exon of c-myc oncogene in squamous cell carcinoma of the head and neck]. / Análisis de reordenamientos en el primer exón del oncogén c-myc en carcinomas epidermoides de cabeza y cuello.

Activation of c-myc has been implicated in the origin of different human tumors, and can be produced by diverse mechanisms as amplifications or rearrangements. We examined 55 squamous cell carcinomas of the upper aerodigestive tract for rearrangements involving the first exon of c-myc, which plays a regulatory role in the c-myc expressión. Polymerase chain reaction (PCR) amplification of different fragments of c-myc exon 1 was performed using four oligonucleotide primers that correspond to consecutive sequences from the 5' end of the c-myc exon 1, combined with one oligonucleotide that corresponds to the 3' end. Amplified c-myc PCR products appear as single bands of 580, 495, 417 and 333 base pairs on the electrophoretic analysis. Only in one case a rearrangement involving the first exon of c-myc was found. We concluded that gene rearrangement is not a common mechanism of activation of c-myc in squamous cell carcinomas of the head and neck.

Isolation of nine gene sequences induced by silica in murine macrophages.

Macrophage activation by silica is the initial step in the development of silicosis. To identify genes that might be involved in silica-mediated activation, RAW 264.7 mouse macrophages were treated with silica for 48 h, and a subtracted cDNA library enriched for silica-induced genes (SIG) was constructed and differentially screened. Nine cDNA clones (designated SIG-12, -14, -20, -41, -61, -81, -91, -92, and -111) were partially sequenced and compared with sequences in GenBank/EMBL databases. SIG-12, -14, and -20 corresponded to the genes for ribosomal proteins L13a, L32, and L26, respectively. SIG-61 is the mouse homologue of p21 RhoC. SIG-91 is identical to the 67-kDa high-affinity laminin receptor. Four genes were not identified and are novel. All of the mRNAs corresponding to the nine cloned cDNAs were inducible by silica. Steady-state levels of mRNAs in RAW 264.7 cells treated with various macrophage activators and inducers of signal transduction pathways were determined. A complex pattern of induction and repression was found, indicating that upon phagocytosis of silica particles, many regulatory mechanisms of gene expression are simultaneously triggered.