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BACKGROUND: Bicarbonate reabsorption in the kidney proximal tubule is predominantly mediated via the apical Na+/H+ exchanger (NHE-3) and basolateral Na+: HCO(-3) cotransporter (NBC-1). The purpose of these studies was to examine the effects of Na+ load and altered acid-base status on the expression of NHE-3 and NBC-1 in the kidney. METHODS: Rats were placed on 280 mmol/L of NaHCO(3), NaCl, or NH(4)Cl added to their drinking water for 5 days and examined for the expression of NHE-3 and NBC-1 in the kidney. RESULTS: Serum [HCO(-3)] was unchanged in NaHCO(-3) and NaCl-loaded animals versus control (P> 0.05). However, a significant hyperchloremic metabolic acidosis was developed in NH4Cl-loaded animals. A specific polyclonal antibody against NBC-1 recognized a 130 kD band, which was exclusively expressed in the basolateral membrane of proximal tubules. Immunoblot studies indicated that the protein abundance of NBC-1 and NHE-3 in the cortex decreased by 74% (P < 0.04) and 66% (P < 0.03), respectively, in NaHCO(3) loading and by 72% (P < 0.003) and 55% (P < 0.04), respectively, in NaCl loading. Switching from NaHCO(3) to distilled water resulted in rapid recovery of NHE-3 and NBC-1 protein expression toward normal levels. Metabolic acidosis increased the abundance of NHE-3 (P < 0.0001) but not NBC-1 (P> 0.05). CONCLUSIONS: NaHCO(-3) or NaCl loading coordinately down-regulates the apical NHE-3 and basolateral NBC-1 in rat kidney proximal tubule, presumably due to increased Na+ load. We propose that the down-regulation of these two Na+- and HCO(3)-absorbing transporters is, to a large degree, responsible for enhanced excretion of excess of Na+ and alkaline load and prevention of metabolic alkalosis in rats subjected to NaHCO(-3) loading.

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Metastatic lesions of the primary non-small cell lung cancer to the atlas have not been previously described. We report a 66-year-old female patient with the primary non-small cell lung cancer and a metastatic lesion to the atlas. Clinicopathologic, radiologic features and treatment of this tumor are discussed.

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The present study was undertaken to determine whether the nm23-H1 gene is expressed in squamous cell carcinoma of the head and neck (SCCHN) and whether the level of nm23-H1 protein or mRNA in cells vary as they progress to a more malignant phenotype. Of the 120 SCCHN studied 54 (45%) stained positively for nm23-H1 protein. Protein expression was significantly higher in more advanced stages of disease. Expression of nm23-H1 was significantly higher in cancer tissues than in normal, adjacent tissue, dysplasia, or carcinoma in situ. The nm23-H1 rate increased with progression of synchronous lesions from dysplasia to carcinoma in situ and finally to carcinoma (P<0.05). Northern blot analyses of tissues with various clinicopathological characteristics also revealed differences in nm23-H1 mRNA expression. When levels of nm23-H1 mRNA were compared to tumor stage, intensity of expression was found to be higher in stages 3 and 4 than stages 1 and 2 (P<0.01). Malignant tumors had a higher level of mRNA nm23-H1 expression than normal or premalignant tissues. The nm23-H1 negative patients survived significantly longer than nm23-H1 positive ones (P<0.05). To study the possible relationship between nm23-H1 gene expression and cell growth rate in tumor cells, the mRNA level in each tumor was compared to proliferative activity. The nm23-H1 gene expression levels were directly related to the [3H]thymidine labeling index in tumor cells (R=0.6681). Our results strongly indicate that the nm23-H1 gene is involved in progression of SCCHN. Together with results obtained on lung cancer, our observations suggest that increased expression of nm23-H1 in cancers of the upper aerodigestive tract may have different implications than elsewhere in the body.

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Relatively little is known about molecular genetic events that participate in the genesis and progression of hemangiopericytoma. In this study, we describe two cases of hemangiopericytoma accompanied by severe hypoglycemia. Tumor cells from patient 1 exhibited insulin-growth factor I (IGF I) and insulin-like growth factor I receptor (IGF IR) mRNA transcripts. Tumor cells from patient 2 exhibited IGF II, IGF IR and IGF binding proteins 1-3 mRNA. Serum from patient 2 contained IGF II, mostly in a large molecular form ("big" IGF II); the IGF II level did not change after the tumor removal. The presence of IGF IR in tumor cells was confirmed by immunoprecipitation with antibodies that recognize human IGF IR subunit (visualized as a 460-kDa band). The hemangiopericytoma cells derived from patient 1 expressed 210000 IGF I receptors/cell. Specific binding of IGF I to the tumor cell membrane fraction was higher in tissue from patient 1, while the tissue of patient 2 showed relatively low IGF I binding. In contrast, IGF II binding was much higher in tissue from patient 2. Both tumor tissues showed positive immunostaining for c-Jun; one tumor showed strong immunostaining for c-Myc, H-Ras and p53, while the other exhibited strong reaction with H-Ras antibodies only. No loss of the heterozygosity at the genes APC, NFI and nm23-H1 loci in tumor tissue obtained from patient 1 was found. In effect, our results suggest multiple molecular genetic changes in hemangiopericytoma -- activation of some oncogenes and the IGF growth factor family. IGF ligands together with IGF IR could be responsible for hypoglycemia and perhaps the transformed phenotype.

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The "bystander effect," produced by ganciclovir-mediated killing of cells transduced with a herpes simplex virus thymidine kinase (HSVtk) gene, defines the cooperative killing of non-HSVtk-transduced cells. In vitro, a major contributor to this phenomenon is metabolic cooperation involving transfer of cytotoxic small molecules between cells via gap junctions. In this study, the bystander effect was assessed in vivo using cells of oral squamous cell carcinoma origin. Mixtures of HSVtk+ and HSVtk- tumor cells were implanted subcutaneously in the left flank of nude mice, and naive HSVtk- cells were implanted subcutaneously in the right flank. When tumors attained a size of 0.5 to 1 cm, the animals were treated with ganciclovir on a daily basis. The tumors comprised of mixed cells in the left flank resolved, consistent with a predicted bystander effect. The naive tumors in the right flank either resolved or became cytostatic showing little further growth compared to controls. Similar results were obtained when naive tumors were grown in both flanks and the tumor in the left flank received intratumoral injection of HSVtk retroviral producer cells or PA317 (HSVtk+) packaging cells, but not parental NIH 3T3 cells. Concomitant treatment with dexamethasone impaired the antitumor effect on the contralateral side. When these experiments were performed in SCID-Beige mice, there was a reduced antitumor effect on the ipsilateral flank and no antitumor response in the contralateral flank. Together with histology of regressing tumors, which showed an infiltration of lymphoid cells, these results are suggestive of an immune-related antitumor response that could account for the distant bystander effect.

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Prostaglandin (PGE(2)) is an inflammatory mediator that plays a critical role in the pathogenesis of periodontal disease. Prostaglandin H synthase (PGHS) a rate-limiting enzyme in PGE(2) biosynthesis exists as two separate isoforms (PGHS-1 and PGHS-2). We have previously demonstrated that both isoforms are generally present in the gingival tissue of periodontitis patients. This study explores in greater detail the variable distribution of each isoenzyme in both inflamed and non-inflamed gingival tissues of patients with periodontitis, and the relationship to adjacent bacteria. Although the positive staining for PGHS-1 was never as intense as for PGHS-2 in the same tissue specimen, either in inflamed or non-inflamed tissues, there was strong staining for both isoenzymes in the epithelium. The keratin layer did not stain. Non-keratinizing crevicular and junctional epithelium contained both isoenzymes through their full thickness in both inflamed and non-inflamed tissues. Pronounced staining of PGHS-2 was evident in the epithelia adjacent to Gram-positively stained organisms. In non-inflamed tissue, PGHS-1 and PGHS-2 were particularly evident in the spinous cell layer; however, fewer of the fibroblasts, endothelial cells, and resident mononuclear inflammatory cells stained positively for PGHS-1 as compared to PGHS-2, but this was less apparent in the inflamed tissues. The immunohistochemical staining patterns indicate that both crevicular and gingival epithelium are important sources of prostaglandin production in the gingival tissue of patients with periodontitis and that bacteria entrapped near to these sites may be important in promoting expression of inducible PGHS-2.

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Human glutathione S-transferase pi (GST-pi) may serve as a useful tumor marker because of the high frequency with which it is found in elevated levels in several tumor types. To determine whether GST-pi is useful as an indicator for cancers of the head and neck, expression of GST-pi mRNA was investigated by Northern analysis in this tumor type. Overexpression of GST-pi mRNA was detected in 9 of 36 (25%) primary head and neck squamous cell carcinomas (HNSCCs). When Southern blot analysis was used to examine the relationship between overexpression and amplification of the GST-pi gene, only 3 of 36 tumors (8%) showed GST-pi gene amplification. Thus, gene amplification is not critical to GST-pi mRNA overexpression in HNSCCs. Moderately and poorly differentiated HNSCCs tended to manifest elevated GST-pi mRNA compared with well differentiated tumors (30% for moderately and poorly differentiated tumors versus none of the well differentiated tumors examined). However, there was no significant correlation between GST-% mRNA overexpression and clinical stage, T stage (tumor size), N stage (neck nodal status), pathological nodes, or patient survival.

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Oral cavity cancer is a major health concern worldwide. Despite advances in surgery, radiotherapy and chemotherapy over the past 35 years, there has been no significant enhancement in the survival of oral cavity cancer patients. Improved survival will require identification of reliable prognostic markers that provide a rational basis for assessment of risk for progression. The altered retinoblastoma (RB) gene has been linked to the hereditary retinoblastoma. This gene is defective in several types of human malignancies. The intent of this study was to evaluate the role of the RB gene in oral cavity tumorigenesis and to explore whether or not there is a relationship between the loss of RB protein and each of several clinicopathological parameters in oral cavity carcinomas. We have analysed the expression of the RB gene in four cell lines (J82, ML1, SaOS2 and WERI-RB-1), 182 oral cavity carcinomas (75 T1 and 107 T3 and T4 lesions) and 55 normal tissues adjacent to cancer by means of an immunohistochemical method and Western immunoblotting. The expression of RB protein was then correlated with clinical outcome in the patients with primary tumours. The significantly higher rate of altered RB expression was found in advanced oral cavity tumours (40 of 107; 37%) in comparison with low grade tumours (9 of 75; 7%). In T3 and T4 tumours, RB gene expression did not correlate with presence or absence of lymph node metastasis, degree of differentiation and patient survival. However, in the T1 cohort, poorer survival rate was seen for those patients who had a tumour with loss of RB protein. This study suggests that tumours in which the RB protein was altered were more aggressive than tumours in which the RB protein was present and that loss of RB protein in oral cavity cancer may be a prognostic variable of tumour progression.

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OBJECTIVES: To determine whether the bystander effect demonstrated in vitro for ganciclovir-mediated killing of a herpes simplex virus thymidine kinase (HSV-tk) gene-infected human squamous cell carcinoma is operative in vivo in a nude mouse model. DESIGN: Prospective study in a murine model. INTERVENTION: Human head and neck squamous cell carcinoma tumors were grown as xenografts on the flanks of 20 nude mice. The tumors in the left flank were then infected with the HSV-tk gene. Then, after 48 hours, the animals were treated with intraperitoneal ganciclovir twice daily. Assessment of the tumors on both flanks was performed over a 31-day period. MAIN OUTCOME MEASURES: Resolution of tumors infected with HSV-tk gene in animals treated with ganciclovir; resolution of tumors uninfected with HSV-tk gene on the contralateral flank in animals treated with ganciclovir. RESULTS: Following HSV-tk gene therapy in nude mice, complete resolution of HSV-tk-gene-infected human head and neck squamous cell carcinoma tumors was observed following ganciclovir treatment. Uninfected tumors were also noted to regress, but not completely resolve, in response to intraperitoneal ganciclovir (distant bystander effect). CONCLUSIONS: This study confirms that the local and distant bystander effects exist in this murine model, enhancing the possibility of its role for treatment of human squamous cell carcinoma of the head and neck.

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The objective of this study was to determine whether elevated levels of N-ras correlated with clinicopathological data. Complete clinical data were available on 133 of 481 patients surgically treated for squamous cell carcinoma of the head and neck (SCCHN) who had immunohistochemical data for N-ras. Advanced stages of disease were strongly related to the staining for N-ras in tumour cells (P = 0.0031). The stage of disease was inversely related to duration of survival (P = 0.0017). Initial statistical evaluation revealed an apparent correlation between survival and N-ras staining. However, duration was found to be independent of the level of N-ras. The illusory relationship initially was a result of the confounding effect of the stage of disease.

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Aims-To determine whether mutant p53 alleles harboured by malignant tumours of the oral cavity were also present in previous premalignant lesions at the same site.Methods-Paraffin embedded tumour specimens along with their premalignant counterparts were analysed for p53 alterations using immunohistochemistry, microdissection, polymerase chain reaction amplification, and DNA sequencing.Results-Malignant lesions from five of eight patients showed overexpression of p53 protein by immunohistochemistry. Upon DNA sequencing, two of these five specimens had p53 mutations. Of the five patients whose cancers showed p53 overexpression by immunohistochemistry, three had previous premalignant lesions that also had immunohistochemically detectable p53 protein. However, DNA sequencing showed that none of these three had mutations in the p53 gene. The remaining five premalignant lesions had no immunohistochemically detectable p53 protein.Conclusions-Some premalignant lesions have increased p53 protein which can be detected by staining with antibody to p53. This staining is not caused by mutations in p53 that are found in subsequent tumours at the same site.

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The expression of H-ras, K-ras and N-ras oncogenes was analyzed on frozen sections of squamous cell carcinoma of the head and neck (SCCHN) by immunohistochemistry using anti-ras monoclonal antibodies. Of 22 primary SCCHN, 15 (68%) stained positive for H-ras, 10 (45%) for K-ras and seven (32%) for N-ras. Thirteen specimens (59%) stained positive for at least two anti-ras monoclonal antibodies. The presence of immunohistochemically detectable H-ras, K-ras and N-ras proteins was most frequently associated with an increase in tumor size and later stages of disease (T3 and T4), with no apparent correlation with lymph node involvement, site of occurrence, degree of differentiation, age, sex, or race. Thus, overexpression of members of the ras gene family occurs as a relatively common even in SCCHN and may be an important event in the later stages of tumorigenesis.

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In order to determine whether or not the p53 gene is involved in the malignant transformation of the head and neck carcinoma HNSCC, we have analyzed archival specimens from 527 primary head and neck lesions and 27 corresponding lymph node metastases. Nuclear p53 protein was present in 107 of 190 (56%) dysplasias, 61 of 102 (60%) carcinoma in situ (CIS), and 262 of 493 (53%) carcinomas. The p53 score did not increase significantly with progression of these lesions from dysplasia to CIS and to carcinoma. All 357 normal samples of head and neck tissues were negative. The majority of the 172 sets of premalignant and malignant lesions displayed concordant p53 staining patterns. The staining was incongruous in only six cases. The p53 staining results were congruent in all 27 pairs of primary and metastatic (lymph nodes) tumors. These data strongly suggest that p53 protein could be altered in a very early phase of the head and neck tumorigenesis and is maintained during tumor progression and metastatic spread. Mutations in p53 were examined in 11 cases that exhibited high levels of p53 protein as detected by immunohistochemistry using PAb 1801 MAb. Mutation analysis was performed by direct sequencing of the PCR amplification products of exons 5 through 8, which contain greater than 90% of p53 mutations found in tumors. Three of 11 HNSCC had mutations at codon 130 (C to A), 193 (A to T), 283 (G to C), respectively. No mutations were found in the other 8 samples within the regions examined. However, they may have mutations in unsequenced regions of p53 or may have wild type protein that accumulates for other reasons.

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OBJECTIVES: To evaluate the correlation between the levels of immunohistochemically detectable c-myc protein in squamous cell carcinoma of the head and neck and clinicopathologic prognostic variables utilized in clinical practice. DESIGN: Cohort study. SETTING: University and Veterans Administration medical centers, Cincinnati, Ohio. PATIENTS OR OTHER PARTICIPANTS: Consecutive samples. INTERVENTION: Surgery for squamous cell carcinoma of the head and neck. MAIN OUTCOME MEASURE: Correlation between c-myc expression and tumor size, nodal involvement, clinical disease stage, and degree of differentiation. Hypothesis formulated after data collection. RESULTS: Significant negative correlation between the c-myc levels and the number of metastatic nodes (P = .0001) and clinical stage of disease (P = .05). No correlation with tumor size or degree of differentiation. CONCLUSIONS: Reduction or loss of c-myc oncoprotein might be associated with metastatic lymph node involvement and advanced stages of squamous cell carcinoma of the head and neck. Further studies are needed to substantiate preliminary findings.

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We describe the involvement of HPV31 in a laryngeal carcinoma, a highly unusual anatomic site for this HPV subtype. In situ hybridization with a type-specific HPV probe identified infected tumor cells. Diagnostic Southern blot analysis confirmed that the HPV was type 31, and also revealed that the HPV DNA was episomal. The tumor was passaged in nude mice, and analysis of HPV DNA in the passaged tumor revealed that the viral DNA had persisted and that it had remained episomal. The status of p53 in the tumor was examined by Southern blots and by PCR analysis of a closely linked, highly polymorphic dinucleotide repeat region. There was no apparent allele loss or loss of heterozygosity at p53 or at the locus of another putative tumor suppressor gene at 17p distal to p53. To assess the integrity of the p53 gene in more detail, exons 4 through 11 were amplified by PCR, and the amplified DNA was directly sequenced. No mutations in p53 were observed, suggesting that other mechanisms such as sequestration of p53 by the E6 or E7 products may have contributed to the malignancy.

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OBJECTIVES: To improve the detection of p53 protein in formalin-fixed, paraffin-embedded head and neck tumor tissues. DESIGN: Cohort. SETTING: University and Veterans Administration medical centers. PATIENTS OR OTHER PARTICIPANTS: Retrospective samples. INTERVENTION: Surgery for head and neck carcinoma. MAIN OUTCOME MEASURES: Retrieval of p53 antigen. Hypothesis formulated after data collection. RESULTS: An antigen retrieval method facilitated the unmasking of previously inaccessible p53 antigenic determinants in formalin-fixed, paraffin-embedded tissues. This approach has made possible a much more reliable and sensitive immunohistochemical detection of p53 antigen. The procedure is simple, requiring only microwave heating of tissue sections to 100 degrees C in the presence of a zinc sulfate solution. CONCLUSIONS: Antigen retrieval method in formalin-fixed, paraffin-embedded tissue demonstrated a significant increase in p53 immunostaining.

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P-glycoprotein (Pgp), encoded by the multidrug-resistance gene (MDR1), is an active efflux pump for many structurally diverse lipophilic compounds. Using peroxidase-antiperoxidase immunohistochemistry technique and four anti-Pgp monoclonal antibodies directed against different epitopes of the molecule, we examined the distribution of Pgp in normal human tissues and squamous cell carcinomas of the head and neck. All four antibodies detected Pgp in bronchial cells, mammary ductal epithelium, gallbladder epithelium, epithelia of small and large intestine, bile canaliculi, dermal sweat glands, proximal tubules of kidney, endometrium, trophoblasts, adrenal gland, and capillaries of central nervous system, testis, and papillary dermis. Of the 23 head and neck squamous cell carcinomas, about 60% had detectable Pgp. It is possible that differences noticed between antibodies are due to cross-reactivity to proteins unrelated to MDR1. Care must be taken in interpreting staining results when only one or two monoclonal antibodies are used.

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One established mechanism of multidrug resistance is elevated expression of P-glycoprotein. The expression of P-glycoprotein by immunohistochemistry was examined in squamous cell carcinomas of the head and neck using a newly developed monoclonal antibody, UIC2, which is specific to the human MDR1 gene product and recognizes an external epitope of the protein. P-glycoprotein was detected in 60% of the samples. MDR1 expression was compared with clinical response to chemotherapy in eight patients who received MDR1-dependent drugs and response was accurately predicted in seven (89%) of eight patients. Positive P-glycoprotein staining correlated with the degree of tumor differentiation, but not with other clinical factors. Therefore, analyzing the expression of P-glycoprotein may play a role when planning chemotherapeutic regimens for patients with head and neck cancer and may be an additional prognostic and diagnostic tool in these patients.

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This study examined the relationship between immunohistochemically detectable p53 protein and prognostic factors in squamous cell carcinoma of the head and neck (SCCHN). Twenty-seven tumor specimens were evaluated utilizing a panel of three monoclonal antibodies (MAbs) directed against different epitopes of the p53 protein (PAb 421, PAb 1801, and PAb 240). The overall incidence of p53 protein detection with a panel of MAbs was 78%, which was significantly higher than with any one of the tested antibodies. Comparison of the tumors that were negative for p53 with tumors that stained positive with one or multiple antibodies, however, revealed no statistically significant differences with respect to the stage of disease, metastatic node involvement, size of the primary tumor, or degree of tumor differentiation. The results of our study suggest that levels of p53 protein, although commonly immunohistochemically detected in head and neck tumors, do not correlate with known prognostic factors for SCCHN.

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The c-erbB-2 oncoprotein is a transmembrane protein the presence of which has been associated with poor prognosis in several human neoplasms. However, there has been no comprehensive assessment of its value as a potential prognostic marker in head and neck squamous cell carcinoma. Archival specimens from 93 patients, treated surgically for squamous cell carcinoma of the head and neck between 1981 and 1989, were analyzed by immunohistochemistry using an anti-c-erbB-2 monoclonal antibody; of these, 43 (46%) were positive for c-erbB-2 staining. The majority of stained specimens (41%) displayed staining predominantly at the cell surface, while mixed membrane and cytoplasmic staining was less common (9%). Only 4% shared exclusively cytoplasmic staining. Since the specimens were archival, the cytoplasmic staining is probably a consequence of variable handling and/or fixation at the time of tissue removal. Therefore, only cases exhibiting distinct cell surface membrane staining in more than 10% of tumor cells were regarded as positive. There is a definite association between immunohistochemical detection of c-erbB-2 and head and neck squamous cell carcinoma, since almost half of the tumor specimens manifested detectable c-erbB-2 protein. However, this association could not be extended to a predicted disease progression or outcome, since there was no significant correlation between c-erbB-2 staining and tumor size, stage of disease, histologic differentiation, lymph node status or patient survival.

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