High resolution allelotype of nonfunctional pancreatic endocrine tumors: identification of two molecular subgroups with clinical implications.

A high resolution allelotype for nonfunctional pancreatic endocrine tumors (NF-PETs) has been generated by microsatellite analysis of DNA from 16 frozen cases, each probed with 394 markers. Two subgroups of NF-PETs were found. Seven cases showed frequent, large allelic deletions [loss of heterozygosity (LOH)] with an average fractional allelic loss (FAL) of 0.55, whereas nine cases showed a small number of random losses with a FAL of 0.15. Designated high or low FAL, respectively, these genetic phenotypes showed correlation with the ploidy status: high-FAL tumors were aneuploid, low-FAL were diploid. Chromosomes 6q and 11q showed LOH in >60% of cases. About 50% of cases had losses on 11p, 20q, and 21. Selected LOH analysis on an additional 16 paraffin-embedded NF-PETs confirmed the high frequency of 6q and 11q LOH. The allelotype of NF-PET is markedly different from that of either ductal or acinar tumors of the pancreas as well as from that of functional-PETs. Moreover, whereas deletions involving chromosome 11 also are a feature of functional-PETs, the involvement of chromosome 6q is characteristic of NF-PETs. Survival analysis showed that none of the single chromosomal alterations was associated with outcome, whereas ploidy status is an independent factor adding prognostic information to that furnished by the proliferative index measured by Ki-67 immunohistochemistry.

Renal angiomyolipoma with epithelioid sarcomatous transformation and metastases: demonstration of the same genetic defects in the primary and metastatic lesions.

Angiomyolipoma (AML) is a benign neoplasm that occurs either sporadically or in patients with tuberous sclerosis complex (TSC) and shows frequent allelic losses at chromosome arm 16p. It has been suggested recently that the melanogenesis marker-positive perivascular epithelioid cell (PEC) has been found consistently in AML. The authors report a 50-year-old woman without evidence of TSC affected by classic renal AML containing an area composed of atypical epithelioid cells with the same morphoimmunophenotypic characters of PEC. After 7 years from surgical removal of the lesion, the patient developed a local recurrence and successive lung and abdominal metastases that showed morphologic and immunohistochemical features overlapping those of the epithelioid area of the previously removed AML. Genetic analysis showed that the classic AML and its epithelioid area as well as the pulmonary and abdominal metastases shared the same allelic loss on chromosome arm 16p. Based on these findings, the authors view this case as evidence of a malignant transformation of a classic AML with morphologic, immunophenotypic, and genetic demonstration of its clonal origin.

In vivo protein-DNA interactions on a glucocorticoid response unit of a liver-specific gene: hormone-induced transcription factor binding to constitutively open chromatin.

Transcription from the liver promoter of a 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) gene depends on the presence of glucocorticoids that act via a glucocorticoid response unit (GRU) located in the first intron. The promoter and the GRU are in a constitutively open chromatin configuration. To determine how glucocorticoids would affect factor binding to the GRU in absence of chromatin remodeling, we have used a combination of in vitro DNA-binding assays and in vivo genomic footprinting in rat hepatocytes and hepatoma cells. We found that, in the absence of glucocorticoids, the GRU binds nuclear factor-I (NF-I). Glucocorticoid treatment modified factor binding to the NF-I site and induced the binding of hepatocyte nuclear factor-3 (HNF-3). Transfection assays showed that HNF-3 cooperates with the glucocorticoid receptor in stimulating transcription. In contrast with the lack of effect of glucocorticoids on factor binding to constitutively open GRUs of other genes, HNF-3 binding to the open PFK-2 GRU was hormone-dependent. Therefore, the PFK-2 GRU behaves as a novel type of GRU.

The glucocorticoid receptor regulates the binding of C/EPBbeta on the alpha-1-acid glycoprotein promoter in vivo.

A complex interaction between the Glucocorticoid Receptor (GR), C/EBPbeta, and other transcription factors activate the Alpha-1 Acid Glycoprotein (AGP) promoter in HTC(JZ-1) rat hepatoma culture cells. This effect is mediated by the so-called Steroid Responsive Unit (SRU) of the AGP promoter that contains several binding sites for C/EBP transcription factors, some of which overlap with the Glucocorticoid Responsive Element (GRE). Our in vivo footprinting experiments revealed that the GRE- and the C/EBP-binding sites were already occupied glucocorticoid dependently in HTC(JZ-1) cells 10 min after dexamethasone administration (10(-6) M). Furthermore, local changes in the chromatine structure shown by the appearance of DNAse I hypersensitive sites (HS sites) also took place. These changes were probably dependent on a tissue-specific organization of the chromatine at the SRU because they were not detectable in a different glucocorticoid-responsive cell line (PC12) that did not express AGP. Here, we have also shown that withdrawal of dexamethasone or addition of the anti-glucocorticoid RU486 were able to revert the pattern induced by dexamethasone in vivo. The disappearance of the protected region and the hypersensitive sites, typical of the hormone activated promoter, confirmed the necessity of the GR to be bound by the agonist and the inability of the GR-antagonist complex to bind the DNA. By functional assays, we showed that the occupancy of the SRU by these transcriptional proteins in vivo correlated with the activation of the AGP gene transcription. With these results, we have shown that one of the functions of the GR to activate transcription of the AGP gene is to recruit C/EBPbeta and to maintain it bound at its target DNA sequences (SRU). This process was not accomplished by RU486.

Muscarinic cholinergic and alpha 2-adrenergic receptors in the epithelium and muscularis of the human ileum.

The aim of this study was to characterize the binding and functional properties of muscarinic cholinergic (MCh) and alpha 2-adrenergic receptors in the human ileum to provide insight into pharmacologic strategies for managing urinary and fecal incontinence after bladder and rectal replacement with intestinal segments. MCh and alpha 2-adrenergic binding sites were characterized in the epithelium and muscularis of eight human ileal segments with 3H-N-methylscopolamine and 3H-rauwolscine, respectively. The dissociation constant for 3H-N-methylscopolamine in the epithelium and muscularis was 0.32 +/- 0.07 nmol/L and 0.45 +/- 0.10 nmol/L, respectively (p = 0.32). The MCh receptor content was approximately eightfold greater in the muscularis compared with the epithelium (p = 0.008). The dissociation constant for 3H-rauwolscine in the muscularis and epithelium was 2.55 +/- 0.42 nmol/L and 2.03 +/- 0.19 nmol/L, respectively (p = 0.29). The alpha 2-adrenoceptor density was twofold greater in the epithelium compared with the muscularis (p = 0.05). Noncumulative concentration-response experiments were performed with carbachol, an MCh agonist, and UK-14304, a selective alpha 2-adrenergic agonist. The epithelium did not contract in the presence of high concentrations of carbachol and UK-14304. The muscularis preparations were responsive only to carbachol. The muscularis contains primarily MCh receptors mediating smooth muscle contraction. The alpha 2-adrenoceptors are localized primarily to the epithelium and may regulate water secretion in the intestine. The distribution and functional properties of ileal MCh and alpha 2-adrenergic receptors provide a theoretic basis for the treatment of incontinence after bladder and rectal replacement with intestinal segments.

Genetic alterations in primary mediastinal B-cell lymphoma: an update.

Primary mediastinal B-cell lymphoma (PMBL) is a distinct clinical entity among non-Hodgkin's lymphoma. The malignancy has received little attention from a standpoint of basic research due in part to its rarity. However, based on recent studies consistent trends are beginning to emerge regarding the molecular and chromosomal alterations commonly observed in this disease. By both CGH and AP-PCR, genetic gains involving chromosomes 2, 5, 7, 9p, 12, and Xq are among the most frequently observed events. From a molecular standpoint, alterations in the c-myc, p16(INK4) and p53 genes have been observed in up to 30% of cases. This information along with the well-established histological, immunological, and clinical features should convince the few remaining disbelievers that PMBL is a distinct pathological entity among non-Hodgkin's lymphomas.

[Techniques of autologous transfusion in hip surgery: a retrospective study]. / Techniques de transfusion autologue dans la chirurgie de la hanche: étude rétrospective.

A retrospective study was carried out on 99 patients undergoing hip surgery to evaluate the efficacy of three autotransfusion techniques on sparing of homologous blood. Preoperative normovolemic hemodilution (HDN), delayed autotransfusion (ATD) and recuperation of blood lost during surgery (RSPO) were studied. Patients were divided into 5 groups: group HDN, group ATD, group ATD + HDN, group RSPO, and the control group. The hematocrit was followed intra and postoperatively within these five groups, and mean values homologous blood transfusions calculated for each group. Comparison of mean values was performed using variance analysis (p less than 0.02). The association of delayed autotransfusion and preoperative hemodilution was most effective in sparing homologous blood (0.11 +/- 0.3 unit, p less than 0.02): however these results were obtained with a substantial drop in hematocrit which fell well below the 30% minimal accepted value at the end of the operation. There was no significant difference between delayed autotransfusion (0.35 +/- 0.8 unit) and preoperative hemodilution (0.7 +/- .01 unit) in sparing homologous blood. Hematocrit values with delayed autotransfusion were greater than these obtained with preoperative hemodilution. The difference was significant at the beginning of the operation (p less than 0.001), but not significant at the end. Recuperation of blood lost during surgery was least effective in sparing homologous blood (1 +/- 1.2 unit, p less than 0.02). Because of the moderate decrease in hematocrit, there are very few contraindications to delayed autotransfusion. Recuperation of blood lost during surgery should be associated with another autotransfusion technique.(ABSTRACT TRUNCATED AT 250 WORDS)

The efficacy of transurethral resection of the prostate in men with moderate symptoms of prostatism.

Transurethral resection of the prostate represents the operation most commonly performed by urologists in the United States. The objective of this study was to determine the efficacy of transurethral prostatectomy in men with moderate symptoms of prostatism. The efficacy parameters evaluated included peak urinary flow rate, obstructive and irritative symptom scores, and the global assessment by the patient of the voiding symptomatology. The mean peak urinary flow rate improved 108% after transurethral prostatectomy, and the mean obstructive and irritative symptom scores decreased 88 and 65%, respectively. The observed changes in urinary flow rates and symptom scores were statistically and clinically significant. Over-all, 84% of the patients indicated that the voiding symptoms were markedly improved after prostatic resection. Baseline urodynamic parameters were of no value for prediction of postoperative outcome. Several investigators recently reported their clinical experience with various pharmacological approaches for the treatment of symptomatic benign prostatic hyperplasia. Over-all, the degree of improvement in urinary flow rates and symptom scores achieved after transurethral prostatectomy exceeds all other therapeutic options that presently are available for the treatment of benign prostatic hyperplasia.

Corrective procedures for penile shortening due to Peyronie's disease.

Peyronie's disease is a poorly understood scarring condition of the tunica albuginea that often causes pain and deformity of the penis. When Peyronie's disease is present predominantly unilaterally, bending of the penis to 1 side occurs. With bilateral or circumferential disease the tunica albuginea does not expand, and penile shortening and/or narrowing occurs. We performed unilateral or bilateral penile lengthening procedures on 22 patients with penile shortening due to Peyronie's disease who refused a procedure, such as tucks or the Nesbit operation, that might result in further penile shortening. Five men underwent incision of the plaque and dermal patch without implantation of a penile prosthesis, while 8 underwent penile implantation plus incisions in the tunica albuginea without patches, and 5 underwent circumferential incision of the tunica albuginea and its septum with patching and placement of a penile implant. (Average length gain with circumferential incision was 1.5 inches with this procedure.) There were 18 patients available for followup: 1 had penile skin slough secondary to a compression dressing, 2 required removal of the implant and replacement at a later date, and 2 had progressive penile shortening after dermal patch requiring subsequent prosthesis implantation. Penile lengthening procedures to correct functionally significant penile shortening can be performed successfully, although significant complications were experienced.

In vivo modification of major histocompatibility complex class II DRA promoter occupancy mediated by the AIR-1 trans-activator.

RJ 2.2.5 is a human B cell mutant derived from the Burkitt lymphoma Raji cell which is defective in the AIR-1 locus function. This locus encodes a transcriptional trans-activator required for the constitutive expression of major histocompatibility complex (MHC) class II genes. Here we show, by in vivo DNase I footprinting, that the AIR-1 locus defect correlates with changes in the DRA promoter occupancy. Interestingly, reexpression of human MHC class II genes in RJ 2.2.5 x mouse spleen cell hybrids is associated with partial reversion of DRA promoter occupancy to the Raji pattern. DRA promoter occupancy in other class II-negative B cell lines, derived from patients with bare lymphocyte syndrome, is drastically different from the one observed in RJ 2.2.5 and Raji cells. Moreover, the use of the DNase I as an in vivo footprinting agent reveals that the patients' cell lines do not display a completely "bare promoter" as previously reported using dimethyl sulfate as the footprinting agent. Thus, the use of DNase I allowed us, for the first time, to correlate the AIR-1 locus defect with class II promoter occupancy alterations and distinguish these alterations from the ones observed in phenotypically similar but genetically distinct MHC class II-negative cells.

Primary tumors of the trachea. Results of radiation therapy.

From 1959 to 1986, 24 patients with primary malignant tumors of the trachea received radiotherapy as all or part of treatment. Common presentations included respiratory symptoms in 20 patients and hemoptysis in 15. Thirteen patients had squamous carcinomas with undifferentiated and adenoid cystic cancers in five and four patients, respectively. Overall actuarial survival was 45% at 1 year, 25% at 5 years, and 13% at 10 years. Survival was significantly correlated to histologic type (adenoid cystic versus squamous, P less than 0.03), but not to tumor extent or to patient age or sex. Local control was attained in 10 of 24 patients overall and was more frequent for patients with tumors localized to the trachea and for patients who were treated with combined surgery and radiotherapy. For the 18 patients treated with radiotherapy alone, complete response (CR) was seen to be significantly (P less than 0.001) related to dose: six of seven (86%) patients receiving greater than or equal to 6000 cGy attained CR versus one of 11 (9%) receiving less than 6000 cGy. Three patients developed complications related to radiotherapy. Radiotherapy can provide durable local control of localized tracheal tumors and should be considered for medically inoperable patients with localized tumors and for patients with high risk of recurrence after resection.

Two remote glucocorticoid responsive units interact cooperatively to promote glucocorticoid induction of rat tyrosine aminotransferase gene expression.

Tyrosine aminotransferase (TAT) gene transcription is specifically activated by glucocorticoid hormones in liver cells. This regulation involves a glucocorticoid responsive region located 2,500 bases upstream from the transcription start site of the rate gene. By transient transfection of TAT-CAT fusion genes into a rat hepatoma cell line expressing the TAT gene we found that this region promotes only 30% of the glucocorticoid stimulation. We have identified a new cis-acting region far upstream (-5,400) from the transcription start site that is essential to achieve the physiological level of glucocorticoid stimulation of endogenous TAT gene expression. This region corresponds to a tissue-specific DNAse I hypersensitive site which is constitutive despite the fact it possesses a glucocorticoid receptor binding site. It is by itself almost inactive on a promoter but it cooperatively enhances the action of the proximal glucocorticoid responsive region. Its activity requires both the glucocorticoid receptor binding site and its flanking sequences.

In vivo footprinting of rat TAT gene: dynamic interplay between the glucocorticoid receptor and a liver-specific factor.

HNF5, a liver-specific DNA-binding protein, interacts with DNA in a manner that allows DNAase I cleavage in the middle of its recognition sequence. Using this property we have identified in vivo HNF5 bound to its sites within two glucocorticoid-responsive units of the rat tyrosine aminotransferase (TAT) gene. One HNF5-binding site is also a glucocorticoid receptor-binding site; glucocorticoid-dependent HNF5 binding could be detected at this site even though it is incompatible with glucocorticoid receptor binding. HNF5 binds within 10 min of hormone addition, indicating that it participates in transcriptional activation. In the TAT gene glucocorticoid-dependent HNF5 binding occurs where there is glucocorticoid-dependent disruption of nucleosomal structure; constitutive binding occurs in constitutively disrupted regions. These results suggest a hit-and-run mechanism of transcriptional activation by glucocorticoid receptor: the activated receptor binds its target sequence, modifies local chromatin structure, then leaves its site accessible to another factor.

Cell-type specific activity of two glucocorticoid responsive units of rat tyrosine aminotransferase gene is associated with multiple binding sites for C/EBP and a novel liver-specific nuclear factor.

The structures of two remote glucocorticoid responsive units (GRUs) that cooperatively interact to promote cell-type specific glucocorticoid induction of rat tyrosine aminotransferase gene expression have been analyzed. DNAase I footprinting and gel mobility shift analyses reveal a complex array of contiguous and overlapping sites for cell type-specific DNA binding proteins. Apart from the glucocorticoid receptor, two liver-specific nuclear factors possess multiple binding sites in each of these GRUs: C/EBP and a newly identified liver-specific factor: HNF5. C/EBP possesses four binding sites in each GRU; a DNA-binding protein with similar binding specificity has been identified in fibroblasts; this protein could be related to AP-3. HNF5 possesses two binding sites in one GRU and four in the other. There are also HNF5 binding sites in numerous regulatory regions of other liver-specific genes. The interaction of HNF5 with DNA gives a characteristic DNAase I footprint with hypersensitive sites in the middle of the recognition sequence. Some of the C/EBP and HNF5 binding sites overlap in a conserved arrangement.

Pancreatic acinar carcinoma shows a distinct pattern of chromosomal imbalances by comparative genomic hybridization.

Pancreatic acinar cell carcinoma (PAC) is a rare pancreatic tumor for which no information about chromosomal anomalies is available. We examined six primary PACs by comparative genomic hybridization (CGH). All cases showed chromosomal changes. A total of 106 gains and 48 losses was detected. Consensus regions of gain were identified on chromosomes 1, 12, and X: 1q21 in four cases, 1q42 in three cases, 12p11.2 in four cases, and Xq12-21 in three cases. Recurrent losses were found at 16p13.2-p13.1 in three cases and at 16q23 in three cases. To verify these chromosomal imbalances, microsatellite analysis of matched normal and tumor DNA was performed using PCR-amplified markers for chromosomes 1, 12, and 16 in the regions showing nonrandom gains or losses. This analysis showed allelic imbalances in tumor DNA consistent with the CGH profiles. Our CGH study suggests that PAC shows a characteristic pattern of chromosomal alterations, involving gain at 1q, 12p, and Xq and loss of sequences at 16p and 16q. This pattern appears unique among solid tumors and is markedly different from that detected in pancreatic ductal carcinomas by the same technique. This suggests that PAC tumorigenesis involves different molecular pathways than those involved in the more common pancreatic ductal tumors. Genes Chromosomes Cancer 28:294-299, 2000.

Molecular features of primary mediastinal B-cell lymphoma: involvement of p16INK4A, p53 and c-myc.

Primary mediastinal B-cell lymphoma (PMBL) shows chromosome 9p anomalies in 50% of cases. Based on reports that p16INK4A gene, located on this chromosomal arm, is frequently altered in aggressive lymphomas, we analysed for alterations of this gene in 27 cases of PMBL, which were part of a series of 32 PMBL cases that have been characterized for alterations in c-myc, p53, N-ras, bcl-1, bcl-2, bcl-6 and for Epstein-Barr virus (EBV) infection. Four cases showed p16INK4A gene anomalies, including three with promoter methylation and one homozygous deletion. Eight PMBLs showed c-myc rearrangements. Three additional cases showed sequence variations in the c-myc P2 promoter, two of which consisted of the same germline variation involving a novel polymorphic XhoI site. Four tumours contained p53 gene mutations and three had clonal EBV infection. One case had a bcl-6 rearrangement. In conclusion, our study shows that p16INK4, c-myc and p53 alterations occur in 15%, 25% and 13% of PMBLs, respectively. EBV monoclonality was found in 9% of cases, whereas no abnormality was detected in bcl-1, bcl-2 and N-ras. Thus, none of the common genetic aberrations seen in other types of non-Hodgkin's lymphomas appears to be stringently involved in the pathogenesis of this unique lymphoma type.

Divergent evolution in the mechanisms controlling major histocompatibility complex class II gene transcription in mouse and human.

The expression of the major histocompatibility complex (MHC) class II gene family is developmentally regulated and, in general, in a coordinate manner. In this study, we show that the expression of the entire repertoire of human class II genes, otherwise transcriptionally silent in the bare lymphocyte syndrome-derived BLS1 cell line, can be rescued by somatic cell hybridization with normal mouse spleen cells. The analysis of the interspecies cell hybrids revealed a particularly important and unprecedented aspect. A return to the BLS1-like, human MHC class II-negative phenotype due to segregation of mouse chromosomes was accompanied in certain hybrids by loss of IE, but not IA cell surface antigen expression. At the molecular level, this was the result of lack of E alpha-specific mRNA in the presence of E beta-, A alpha- and A beta-specific mRNA. Thus, the mouse trans-acting function operating across species barriers and able to complement the defect of human BLS1 cells diverged in mice to control Ea, but not Eb, Aa and Ab gene expression. These findings suggest that evolutionary pressure has maintained the expression of the MHC class II multigene family under the control of quite distinct species-specific transcriptional mechanisms.

Induction of CIITA and modification of in vivo HLA-DR promoter occupancy in normal thymic epithelial cells treated with IFN-gamma: similarities and distinctions with respect to HLA-DR-constitutive B cells.

In this study, the IFN-gamma induction of MHC class II gene expression in primary cultures of thymic epithelial cells (TEC) was analyzed. This cellular system offers the advantage that MHC class II induction is studied in a "physiologic" cell lineage that, as a result of this expression within the thymus, is thought to participate to the selection and maturation of the T cells. It was found that the MHC class II gene expression was associated with the de novo transcription of the gene encoding the CIITA trans-activator, a crucial MHC class II gene regulatory factor. Furthermore, the anatomy of interaction between the MHC class II DRA promoter and corresponding binding factors was analyzed by in vivo DNAse I footprint. It was found that treatment with IFN-gamma induces changes in the occupancy of the DRA gene regulatory sequences by nuclear factors. The resulting occupancy displays strong similarities with the one observed in the MHC class II-constitutive B cells, represented by both the Burkitt lymphoma line Raji and normal tonsil- derived B cells. However, some peculiar differences were observed between the TEC, either IFN-gamma-induced or not, and the constitutive B cells. These results suggest that both common mechanisms, such as the one mediated by the CIITA trans-activator, and distinct tissue-specific constraints contribute to the transcriptional control of constitutive and IFN-gamma-induced MHC class II gene expression.