Identification of tumor metastasis suppressor region on the short arm of human chromosome 20.

Acquisition of metastatic ability by prostate cancer cells is the hallmark of their lethal trait and outcome. However, the genetic alterations underlying the clinical progression and pathogenesis of prostate cancer are not well understood. Several studies involving loss of heterozygosity (LOH) and comparative genomic hybridization analysis have identified distinctively altered regions on various human chromosomes, and genomic imbalance of chromosome 20 was implicated in progression and recurrence of prostate tumors. To examine the role of chromosome 20 in prostate neoplasms, we introduced this chromosome into highly metastatic rat prostate cancer cells using the microcell-mediated chromosome transfer technique. Introduction of the chromosome resulted in significant suppression of the metastatic ability of the hybrid cells, by as much as 98%, without any interference with the in vivo growth rate or tumorigenicity of primary tumor in SCID mice. Our STS-PCR analysis on 10 hybrid clones indicates that the suppressor activity of chromosome 20 is located in the p11.23-12 region. Further examination of the hybrid clones by experimental metastasis assay and histologic analysis as well as Matrigel invasion assay suggests the involvement of the suppressor region at an early stage of invasion and extravasation. We also investigated the status of the chromosome 20 suppressor region in pathology specimens from human prostate cancer patients and detected the frequent loss of this region in high-grade tumors. These results suggest the presence of a putative suppressor gene on human chromosome 20 that is functionally involved in development of prostate cancer metastases.

Activation of the tumor metastasis suppressor gene, KAI1, by etoposide is mediated by p53 and c-Jun genes.

KAI1 is a metastasis suppressor gene which is capable of inhibiting the processes of tumor metastasis without affecting tumorigenicity per se. We found that etoposide, a topoisomerase II inhibitor, is able to activate the expression of the KAI1 gene in a dose-dependent manner in human prostate cancer cell lines, ALVA, DU145, and PC-3 as well as in human lung carcinoma cell A549. The activation of the KAI1 gene was mainly mediated by the c-Jun gene in the PC-3 and DU145 cell lines, while it was mediated by both p53 and c-Jun genes in the A549 cell line. These results suggest that the augmentation of the KAI1 gene expression is independently controlled by p53 and c-Jun at the transcriptional level in the human cancer cell lines. Furthermore, treatment of these cell lines with etoposide resulted in significant reduction of cellular invasion measured by the Matrigel invasion chamber. Because etoposide has been shown to be effective on advanced prostate cancer when used in combination with other regimens, our results provide further rationale to use this drug as an antimetastatic agent.

Localization of a novel tumor metastasis suppressor region on the short arm of human chromosome 2.

Much of the lethality of malignant neoplasms is attributable directly to their ability to develop secondary growths in organs at a distance from the primary tumor mass, whereas few patients die from their primary neoplasm. Little is known about the molecular mechanism of tumor metastasis, however, which is controlled by a variety of positive and negative factors. In the search for metastasis suppressor genes, we have used the microcell-mediated chromosome transfer method and a rat prostate tumor model in SCID mice. When human chromosome 2 was introduced into the highly metastatic rat prostatic tumor cell, AT6.1, the metastatic ability of this cell was significantly (>99%) decreased in animals. An STS-based PCR analysis for 8 hybrid clones indicates that the suppressor activity is located in the p25-22 region of the chromosome. Furthermore, the AT6.1 cell with human chromosome 2 showed a reduced ability to invade Matrigel, suggesting that the suppressor activity is involved in the step of tumor invasion during the progression of prostate cancer. We have also examined the status of the suppressor region on chromosome 2 in human prostate cancer specimens and found that this region was often lost in high-grade tumors. These results suggest that the putative suppressor gene on chromosome 2 is functionally involved in the progression of human prostate cancer. Genes Chromosomes Cancer 28:285-293, 2000.

Antisense oligodeoxyribonucleotides inhibit the expression of the gene for hepatitis B virus surface antigen.

The effect of a series of antisense oligodeoxyribonucleotide [oligo(dN)] on the expression of the surface antigen (HBsAg) gene of human hepatitis B virus (HBV) was examined using hepatocellular carcinoma cells that contain integrated HBV genomes. Of a number of antisense oligo(dN)s tested, synthetic 15-mers directed at the cap site of mRNA and regions of the translational initiation site of the HBsAg gene were found to be highly effective and inhibited viral gene expression by as much as 96%. The inhibition was specific to the HBsAg gene and appeared to be at the level of translation. These results suggest a therapeutic potential for antisense oligo(dN) in the treatment of patients who are chronically infected with HBV.

Organ-specific distribution of isozymes of 5'-nucleotide phosphodiesterase in mouse.

1. The distribution of isozymes of 5'-nucleotide phosphodiesterase (E.C.3.1.4.1) was examined in various organs of mouse, including liver, spleen, pancreas, heart, lung, kidney, brain and blood. 2. Five isozymes were identified and designated as isozymes I through V. 3. These isozymes are distributed unevenly with respect to the various organs and clear differences were observed in the patterns of distribution among the organs examined. 4. The level of these isozymes was compared in serum of neonate and adult mice, and a higher level of isozyme I and a lower level of isozyme IV were found in neonates compared to adults. 5. These results suggest that each isozyme has different functional roles in individual organs and that these isozymes may be involved in proliferation and development of cells.

Revision of painful distal tip amputations.

From 1968 to 1987, 22 patients were diagnosed with dysfunctioning digits after complete distal digital amputations. Each patient had the proximal portion of the partially amputated phalanx left within the injured digit. On average, 21 months after the initial injury, each patient underwent an excision of the remnant portion of the phalanx which averaged 6 mm (range 1 to 17 mm). All 22 patients reported excellent postoperative results of full function and no residual pain with an average follow up of 9 months. We theorized that localized synovitis produces joint pain related to: 1) nontolerated joint stress loading due to a change in the lever arm length of the amputated phalanx, or 2) inadequate cartilage nutrition owing to lack of stress applied to this joint. Maintaining digital length must be rethought with emphasis placed on painless function. Considering the losses to these patients in terms of time, employment, and money, a distal remnant measuring 4 mm or less should be excised, regardless of the digit, at the time of the injury.

Identification and nucleotide sequence of the minimal replicon of the low-copy-number plasmid pBS2.

The plasmid pBS2 has a low copy number and is endogenous to Bacillus subtilis. The replication of this plasmid depends on the function of most of the host's dna genes including dnaB, which is unique to B. subtilis and is required for both the initiation of chromosome replication and the DNA-membrane association. We have identified the region that is essential for the replication of pBS2 and determined the complete 2279-bp nucleotide sequence of this region. In this region, there are two stretches of sequence homologous to the 18-bp consensus sequence which commonly appears at the origin of replication of plasmids pUB110 and pC194. The entire region contains six sizable open reading frames. Two of them are probably translated. One open reading frame, designated ORF A, coding for 269 amino acids, has significant homology, in terms of amino acid sequence, with the open reading frame of the gene for the Rep U protein of plasmid pUB110. The similarities between pBS2 and other plasmids suggest that the pBS2 may also replicate as a rolling circle, which appears to be the salient feature of a mechanism of replication that is common to small plasmids in gram-positive bacteria.

Triquetral-lunate arthritis secondary to synostosis.

Until recently the problem of painful, symptomatic arthritis of the wrist secondary to congenitally incomplete separation of carpal bones has been infrequently recognized. Five patients with either excessive stress loading or trauma had eight symptomatic wrists with congenitally incomplete separation of the triquetral-lunate joint. Three of these patients had bilateral symptoms. Six of the wrists had been treated by a limited wrist arthrodesis of the triquetral-lunate joint resulting in asymptomatic wrists and improved range of motion. It appears that patients with this congenital condition poorly tolerate stress loading or trauma secondary to deficient intra-articular cartilage formation resulting in a clinical and anatomic state similar to degenerative arthritis. We suggest a limited wrist arthrodesis as definitive treatment for symptomatic congenitally incomplete separation of the triquetral-lunate joint, with possible application in incomplete separation of the other intercarpal joints.

Surgical ligation clip artifacts on CT scans.

Surgical ligation clips of various materials were scanned in a water-filled phantom to determine what artifacts would be produced. Tantalum clips produced severe image degradation, while stainless steel clips produced less severe degradation. Minimal artifacts were observed with titanium clips. Clips molded from an absorbable polymeric material, polydioxanone, were visible on computed tomography (CT) scans and had the least effect on the CT image.

Salmonella mediastinitis--a rare disease.

A case report is presented of a young man who was seen because of pleuritic chest pain and fever. CT cross sectional imaging defined a mediastinal mass. Diagnosis of acute mediastinitis due to Salmonella java was made from culturing material at mediastonotomy.

The value of radiographic and computed tomography in the staging of lung carcinoma.

A prospective double-blind study was undertaken to compare computed tomography (CT) and conventional radiographic tomography (RT) in the staging of lung carcinoma. Seventy-five patients had CT and RT of the mediastinum and hilum prior to operation. The presence or absence of metastasis to lymph nodes documented at the time of operation was the standard applied to the studies. CT correctly predicted the presence or absence of mediastinal lymphadenopathy in most cases (sensitivity 91%, specificity 94%), while RT was less helpful (sensitivity 61%, specificity 86%). Metastatic mediastinal lymph nodes in those patients with false negative CT and RT studies averaged only 0.8 cm in diameter, probably accounting for the negative radiographic findings. Both CT and RT had poor predictive values in detecting hilar lymphadenopathy (sensitivity 73% and 47%, specificity 87% and 72%, respectively). The predictive value of CT in the evaluation of mediastinal lymphadenopathy equaled that of mediastinoscopy or mediastinotomy. When CT of the mediastinum demonstrates no lymphadenopathy, invasive staging can be deferred for definitive thoracotomy. Since false positive values were seen with both CT and RT scans of the mediastinum (4% and 8%, respectively), invasive staging will still be necessary in those patients with positive studies.

Immobilized oligodeoxynucleotides as probes of the DNA-binding sites of mouse steroid holoreceptors.

Steroid hormone receptors are DNA-binding proteins with distinct domains for the steroid ligand and for polydeoxyribonucleotides. The characteristics of the polynucleotide-binding sites of the mouse liver dexamethasone receptor, kidney testosterone and estradiol receptors, and uterine estradiol receptor were examined by using oligodeoxynucleotides covalently linked to cellulose as probes. Only dexamethasone receptor binding to oligodeoxynucleotides was dependent upon activation by increased salt concentrations or temperature. The dexamethasone and estradiol receptors required a distinct concentration of KCl for maximal binding to oligodeoxynucleotide-cellulose. In contrast, binding of mouse kidney testosterone receptor was not stimulated by KCl although it was decreased at 0.2 M KCl. The nucleotide affinities of the mouse receptors generally followed the order of binding to oligo(dG)- greater than oligo(dT)- greater than oligo(dC)- greater than oligo(dA)-cellulose. However, the liver dexamethasone receptor bound to oligo(dT)- more avidly than to oligo(dC)-cellulose, while neither the testosterone nor the estradiol receptors clearly differentiated between the oligodeoxypyrimidine celluloses. Although the binding of the kidney testosterone receptor was poorest of those studied, it bound to oligo(dA)-cellulose relatively more efficiently than did the other steroid receptors. Thus, differences were revealed in the requirements for activation, added salt for binding, and relative binding efficiencies to the ligands among different steroid receptors from the same tissue and among the same steroid receptors from different tissues.

Effect of pyridoxal 5'-phosphate on oligodeoxynucleotide binding of mouse uterine cytosol estradiol-receptor complexes.

Pyridoxal-5'-phosphate, an inhibitor of DNA binding of glucocorticoid-, progesterone-, and estrogen-receptor complexes, also blocks the interaction of mouse uterine cytosol estradiol-receptor complexes with oligodeoxynucleotides covalently linked to celluloses. Other derivatives of pyridoxine were ineffective. The inhibition was irreversible only after borohydride reduction. Experiments with three oligodeoxynucleotides showed a 4-fold range of differences in the pyridoxal-5'-phosphate concentrations needed to inhibit 50% of the binding: oligo(dG)-cellulose, 3.1 mM; oligo(dT)-cellulose, 1.5 mM; and oligo(dC)-cellulose, 0.8 mM. Inhibition of binding occurred more rapidly with the oligodeoxypyrimidines than with oligo(dG)-cellulose. Pyridoxal-5'-phosphate was competitive with the oligodeoxynucleotides in its mode of inhibition, suggesting the polynucleotide-binding domain as a site of action. These data indicate a microheterogeneity of oligodeoxynucleotide-binding sites in the estradiol-receptor complex which is reflected in sensitivity to pyridoxal-5'-phosphate.

Diethylpyrocarbonate inhibition of estradiol receptor binding to oligonucleotides.

Exposure of calf uterine estradiol-receptor complexes to diethylpyrocarbonate (ethoxyformic anhydride) at pH 6.3-6.5 results in a decrease in the ability of the receptor to bind to oligodeoxyribonucleotides. The inhibition of binding to oligodeoxypyrimidines is greater than the inhibition of binding to oligodeoxyguanylate. The inhibition by 6.6 mM diethylpyrocarbonate is complete within 10 min at 4 degrees C. Addition of equimolar quantities of histidine or imidazole prior to exposure to diethylpyrocarbonate prevents subsequent inhibition of oligodeoxyribonucleotide binding. In comparison to histidine, other amino acids tested were deficient in this ability. Diethylpyrocarbonate modification of the receptor causes complete loss of oligodeoxyribonucleotide binding activity at times when there is a loss of less than 20% of bound steroid. Pyridoxal 5'-phosphate treatment of receptor does not prevent subsequent modification by diethylpyrocarbonate, suggesting that the site of reaction is not an essential lysine of the DNA-binding domain. Treatment of the ethoxyformylated receptor with 0.45 M hydroxylamine results in recovery of 70% of the receptor's oligonucleotide-binding ability. The time course of the reaction of diethylpyrocarbonate with the estradiol receptor and the demonstration of hydroxylamine reversal of inhibition suggest that histidine is involved in the binding of estradiol receptor to oligodeoxyribonucleotides.

Computed tomography in dissection of the thoracic aorta.

Eleven patients presenting with thoracic aortic dissection were studied by computed tomography (CT). CT was usually performed with knowledge of the angiographic diagnosis. In eight cases, CT was definitive. This group included all six patients who received intravenous contrast material by bolus infusion. An algorithmic approach to patients with possible aortic dissection is suggested.

Pancreatic pseudocyst simulating dilated biliary duct system on computed tomography.

A case of pseudocyst simulating the biliary ductal system is reported. Several CT findings indicate that the biliary system was not involved: (a) no dilatation of the more proximal intrahepatic biliary radicles; (b) the gallbladder was not distended; (c) lucency extending to the left at the level of the pancreas; (d) a position more medial and anterior than anatomic for the distal common bile duct.