Differential expression of S19 ribosomal protein, laminin-binding protein, and human lymphocyte antigen class I messenger RNAs associated with colon carcinoma progression and differentiation.

Three complementary DNA encoding S19 ribosomal protein (S19), laminin-binding protein (LBP), and HLA class I (HLA-I) genes were isolated from a colon tumor-enriched subtraction library. To evaluate this mRNA expression, surgically removed colon tumors as well as matched normal tissue and human colon carcinoma cell lines showing various differentiation states, anchorage dependence, and proliferation states were examined by Northern blot analysis. The mRNA level of S19 mRNA (0.6 kilobase) was higher in primary colon carcinoma tissue than in matched normal colon tissue in 5 of 6 cases. In 2 of 4 cases, the expression of LBP mRNA (1.2 kilobases) was higher in carcinoma than in normal tissue. In 12 human colon cell lines, the level of LBP mRNA was higher in poorly differentiated cells. On the other hand, HLA-I mRNA (1.7 kilobases) was higher in well-differentiated cells. Although the S19 mRNA was expressed in both well- and poorly differentiated cells, a concomitant increase with tumor progression was observed in two pairs of cell lines derived from the same patients (SW480 and SW620; COLO201 and COLO205). Anchorage dependence of butyrate-treated HT29 colon carcinoma cells was correlated with lower levels of S19 and LBP mRNAs and higher levels of HLA-I mRNA expression compared with untreated cells. While the expression of S19 and LBP mRNAs was not changed due to cell growth states, HLA-I mRNA levels were found to be low in proliferating HT29 cells but highly induced in contact-inhibited cells. In summary, therefore, high expression of S19 and LBP combined with low expression of HLA-I were well correlated with colon carcinoma cells of higher malignant potential.

Identification and characterization of genes associated with human hepatocellular carcinogenesis.

Eight cDNAs encoding galectin 4 (Gal-4), UGT2B4 (UDP-glucuronosyltransferase), ribosomal phosphoprotein P0 (rpP0), dek, insulin-like growth factor binding protein (IGFBP) 1, vitronectin, retinoic acid-induced gene E (RIG-E), and CYP3A4 (cytochrome P450 nifedipine oxidase) were identified as differentially expressed genes between human hepatocellular carcinoma (HCC) and matched nontumorous liver tissues. Higher levels of UGT2B4, rpP0, dek, vitronectin, Gal-4, and IGFBP-1 mRNAs combined with a lower level of RIG-E mRNA were observed in at least four of five primary HCCs compared to matched nontumorous liver tissues. Furthermore, a pathological study suggested that the levels of UGT2B4, rpP0, dek, and vitronectin increased and the level of RIG-E decreased with the histological grading. On the other hand, the expression of CYP3A4 mRNA and CYP3A7 (P-450 Fla) mRNA, a transcript found in the fetus and highly homologous to CYP3A4, was higher in all nontumorous liver and some of the carcinoma tissues from five HCC patients, whereas it was significantly lower in normal liver tissues from two non-HCC patients. The examination using HCC cell lines HuH-7 and HepG2 under different growth conditions suggested that the expression of dek mRNA was growth-associated. In contrast, the expression of Gal-4, UGT2B4, IGFBP-1, and RIG-E mRNAs was regulated in a cell density-dependent manner: the levels of Gal-4, UGT2B4, and IGFBP-1 were undetectably low, whereas the level of RIG-E was high in rapidly proliferating, subconfluent HCC cells in 10% serum; however, the expression levels were reversed in dense, overcrowded cultures. In addition, IGFBP-1 and Gal-4 mRNAs were also induced by reducing the serum concentration to 0.1%. We also demonstrated that sodium butyrate, an inducer of differentiation, up-regulated and down-regulated RIG-E and dek mRNAs, respectively, in a dose-dependent manner in HuH-7 cells, supporting, in part, our pathological observation. In summary, therefore, high expression of Gal-4, UGT2B4, rpP0, dek, IGFBP-1, and vitronectin, together with low expression of RIG-E, was correlated with the malignant potential of HCC. CYP3A4 and CYP3A7 could be induced in HCC-bearing livers. These transcripts are differentially regulated depending on cell-cell contact, serum growth factors, growth and differentiation status, and/or other mechanisms in premalignant and malignant liver cells.

The S29 ribosomal protein increases tumor suppressor activity of K rev-1 gene on v-K ras-transformed NIH3T3 cells.

The human S29 ribosomal protein (S29 rp) cDNA has been isolated from differential hybridization screening of a colon carcinoma cDNA library. Northern blot analysis showed that the level of S29 rp mRNA was higher in undifferentiated HT29 human colon carcinoma cells than in a morphologically differentiated subclone under the same growth condition. Furthermore, the level of S29 rp mRNA was downregulated in rapidly proliferating HT29 cells, as compared to the contact inhibited cells. Interestingly, the amount of Krev-1 mRNA was inversely correlated with respect to the amount of S29 rp mRNA in these cells. To examine a functional link between S29 rp and Krev-1 protein, we co-transfected the expression vectors containing wild-type or mutant S29 rp and mutationally activated Krev-1(63E) cDNAs into the v-Ki-ras-transformed NIH3T3 (DT) cells, and observed the induction of flat revertants. Krev-1(63E) induced a certain amount of flat colonies, while S29 rp alone also induced flat colonies at low frequencies. Interestingly, revertant-inducing activity of Krev-1(63E) was significantly enhanced by S29 rp. We have also demonstrated that a zinc finger-like domain of S29 rp indeed has a zinc binding activity and a derivative, S29 rp(ms), which was unable to bind zinc ion but still retained revertant inducing activity by itself, could not functionally interact with Krev-1(63E) protein.

Enhanced expression of mRNAs of antisecretory factor-1, gp96, DAD1 and CDC34 in human hepatocellular carcinomas.

To identify differentially expressed genes in hepatocarcinogenesis, we performed differential display analysis using surgically resected hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues. We identified four cDNA fragments upregulated in HCC samples, encoding antisecretory factor-1 (AF), gp96, DAD1 and CDC34. Northern blot analysis demonstrated that these mRNAs were expressed preferentially in HCCs compared with adjacent non-tumorous liver tissues or normal liver tissues from non-HCC patients. The expression of these mRNAs was increased along with the histological grading of HCC tissues. These mRNA levels were also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. We also demonstrate that sodium butyrate, an inducer of differentiation, downregulated the expression of AF and gp96 mRNAs, supporting in part our pathological observation. Immunohistochemical analysis revealed that gp96 and CDC34 proteins were preferentially accumulated in cytoplasm and nuclei of HCC cells, respectively. Overexpression of these genes could be an important manifestation of HCC phenotypes and should provide clues to understand the molecular basis of hepatocellular carcinogenesis.

The role of Ets family transcription factor PU.1 in hematopoietic cell differentiation, proliferation and apoptosis.

The PU.1 gene encodes an Ets family transcription factor which controls expression of many B cell- and macrophage-specific genes. Expression of the gene is critical for development of lymphoid and myeloid cell lineages, since PU.1-deficient mice exhibit defects in the development of these cell lineages. The PU.1 gene is identical to the Spi-1 gene isolated from common proviral integration sites in Friend virus-induced murine erythroleukemia (MEL), and deregulated expression of the gene is believed to be an essential step of the disease. We recently demonstrated that overexpression of PU.1 inhibits erythroid differentiation of MEL cells induced with the differentiating agent DMSO. We also noticed unexpectedly that overexpression of PU.1 together with DMSO induces marked growth arrest and apoptosis in MEL cells, supporting the notion that some oncogenes induce growth inhibition and apoptosis rather than cell proliferation and transformation under specific circumstances as shown with the c-myc gene. In this review, the role of PU.1 in hematopoietic cell differentiation, proliferation and apoptosis is described and the possible molecular mechanisms of PU.1-induced effects in MEL cells are discussed.

Characteristics of frequency content of atrial signal-averaged electrocardiograms during sinus rhythm in patients with paroxysmal atrial fibrillation.

To clarify the characteristics of the frequency content of atrial signal-averaged electrocardiograms (ECGs) during sinus rhythm in patients with paroxysmal atrial fibrillation, P wave-triggered signal-averaged ECGs were recorded in 28 patients with and 34 control patients without paroxysmal atrial fibrillation. Fast Fourier transform analysis was performed on the 100-ms segment starting 75 ms before the end of the P wave. An area ratio (AR50) was calculated by dividing the area under the spectrum curve between 20 and 50 Hz, multiplied by 100, by the area between 0 and 20 Hz. Magnitude ratios (MR20, MR30, MR40 and MR50) were calculated by dividing the magnitude at 20, 30, 40 and 50 Hz, respectively, multiplied by 100, by the maximal magnitude of the entire signal. AR50 was significantly greater in patients with than without paroxysmal atrial fibrillation (62.3 +/- 34.2 vs. 42.4 +/- 18.4). MR20 and MR30 were also significantly greater in patients with than without paroxysmal atrial fibrillation (MR20 76.1 +/- 15.2 vs. 60 +/- 20.2; MR30 41 +/- 18.8 vs. 26.6 +/- 14.4), although no significant differences in MR40 or MR50 were observed between the two patient groups. The difference in MR30 between groups remained significant even after taking into account the presence of organic heart disease. It is concluded that, irrespective of the presence of organic heart disease, the terminal portion of the P wave contained significantly more components in the 20- to 50-Hz range, especially around 30 Hz, in patients with than in patients without paroxysmal atrial fibrillation. These results suggest that frequency analysis could characterize atrial signal-averaged ECGs of patients at risk for paroxysmal atrial fibrillation.

Which subgroup of patients with dilated cardiomyopathy would benefit from long-term beta-blocker therapy? A histologic viewpoint.

OBJECTIVES: The purpose of this study was to elucidate whether the effectiveness of long-term beta-blocker therapy could be predicted before this therapy is started. BACKGROUND: Long-term beta-blocker therapy has recently been reported to provide a favorable effect in treatment of congestive heart failure due to dilated cardiomyopathy. METHODS: Several measurements including histologic variables before administration of metoprolol were retrospectively compared among 18 good responders (showing improvement of at least one New York Heart Association functional class or an increase in ejection fraction > or = 0.10 12 months after drug administration) and 12 poor responders without such improvement. RESULTS: Although there were no significant differences between the two groups in age, gender, functional class, heart rate, blood pressure, pulmonary capillary wedge pressure, cardiac index, left ventricular end-diastolic dimension and ejection fraction, percent fibrosis estimated by the point-counting method in endomyocardial biopsy specimens was significantly lower in good than in poor responders (7.6 +/- 5.7 vs. 14.2 +/- 9.7%, p < 0.05). Moreover, when the types of fibrosis were classified as interfascicular and intercellular by the dominance of counted points, there were 13 cases of interfascicular fibrosis and 5 cases of intercellular fibrosis in good responders and 1 case of interfascicular fibrosis and 11 cases of intercellular fibrosis in poor responders (p < 0.001, sensitivity 72%, specificity 91%, predictive accuracy 80%). These results suggest that improvement with long-term beta-blocker therapy may be more likely to occur in patients with less myocardial fibrosis, with interfascicular fibrosis the dominant type. CONCLUSIONS: The extent and type of fibrosis may be important factors in the prediction of the effectiveness of long-term beta-blocker therapy for dilated cardiomyopathy.

Serial analysis of gene expression in HIV-1-infected T cell lines.

The gene expression profile of the HIV-1 infection state was analyzed in the human T cell line MOLT-4. Using the serial analysis of gene expression (SAGE) method, a total of 142¿ omitted¿603 SAGE tags were sequenced and identified, representing 43¿ omitted¿581 unique mRNA species. Comparison of expression patterns revealed that 53 cellular genes were differentially expressed upon HIV-1 infection. Northern blot and RT-PCR analyses confirmed the altered expression of the genes in both MOLT-4 and MT-4 cells. Up-regulated genes were mainly composed of transcription factors and genes related to T cell activation, whereas down-regulated genes were comprised of mitochondrial proteins, actin-related factors and translational factors. These findings indicate that persistent T cell activation, which may accelerate HIV-1 replication, and the disruption of cellular housekeeping genes including those involved in anti-apoptotic systems, may play an important role in HIV-1-induced pathogenesis.

Identification and characterization of differentially expressed mRNAs in HIV type 1-infected human T cells.

We used a novel differential display (DD) technique to identify host factors involved in virus replication, pathogenesis, and host response in HIV-1-infected T cells. Thirteen cDNA fragments differentially expressed in HIV-1NL4-3-infected MT-4 cells prior to the occurrence of specific apoptotic cell death were sequenced and identified. Two of seven elevated genes were identical to HIV-1 sequences and the other five were MIP-1alpha, ACTE-III, CD11c, arginase I, and CCR5. The six downregulated genes included prothymosin-a, Jaw-1, proteasome subunit XAPC7, splicing factor 9G8, GA17 protein, and an unknown mRNA. Northern blot and RT-PCR analyses confirmed the altered gene expressions in MT-4 cells as well as in another T cell line, MOLT-4. We also revealed that the amount of MIP-1alpha in culture supernatant of HIV-1-infected cells was increased by more than 15-fold relative to control cells, and the expression of its receptor CCR5 was cooperatively upregulated on the surface of these cells. Furthermore, the upregulation of CD11c after HIV-1 infection was slightly inhibited by blocking the MIP-1alpha-mediated signal transduction. These results indicate that genes altered on HIV-1 infection may be mutually organized and play an important role in HIV-1-induced pathogenesis.

Unresponsiveness of both non-rearranged and rearranged c-myc to serum stimulation in a mouse plasmacytoma S194.

Expression of non-rearranged and rearranged c-myc in a mouse plasmacytoma cell line S194 cultured in different serum concentration or temperature was examined. Exponentially growing S194 cells expressed a high level of rearranged c-myc and a low level of non-rearranged c-myc mRNAs. The levels of the two c-myc mRNAs were nearly the same in the cells in which growth was suppressed by lowering serum concentrations or incubation temperature in culture medium. Furthermore, even when serum-deprived S194 cells resumed growth following serum restimulation, the induction of non-rearranged c-myc mRNA, observed in a mouse T-cell lymphoma cell line BW5147, was not demonstrated. In contrast to the unchanged c-myc expression, the level of N-ras mRNA was related to changes in cell growth rate induced by changes in serum concentration in S194 cells. These results suggest that not only the rearranged c-myc but also the non-rearranged c-myc is unresponsive to serum stimulation and temperature changes, even though cell growth rate is markedly changed.

Chromosomal alterations in permanently differentiated ht29 colon-carcinoma cells induced with sodium-butyrate.

We have established differentiated subclones from sodium butyrate-treated HT29 colon carcinoma cells. Two subclones, CIII and CVII, grew as tightly-compacted colonies and showed polarized confluent monolayers in vitro. CIII cells formed domes in vitro, a morphological characteristic of columnar absorptive epithelium. Besides these differentiated characteristics, CIII and CVII cells showed lower tumorigenicity than parental HT29 cells. Karyotypic analysis indicated that all cells retained chromosomes in the triploid range. However, modal chromosome numbers among 30 metaphases of each clone were concomitantly reduced from 71 (in HT29) to 68 (in CVII) or 67 (in CHI) with the differentiated phenotypes. Our preliminary karyotype analysis has suggested that the number of chromosomes 10q21-qter, 14q22-qter and 16q were decreased in both subclones. To confirm this, DNAs from these cells and another butyrate-induced subclone, CI-1, were analyzed with several DNA markers. Southern blot analysis indicated that parental HT29 cells were polymorphic for the BamHI and TaqI RFLPs detected by urokinase (7 kbp and 1.6 kbp) and D10S25 (2.5 kbp and 2.1 kbp) probes, respectively, both of which are located on chromosome band 10q. In CI-1 and CVII cells, allelic losses of urokinase (1.6 kbp) and D10S25 (2.5 kbp) were observed, respectively. Although both alleles of these markers were conserved in CIII cells, one allele of urokinase (7 kbp) and D10S25 (2.1 kbp) was less representative. Southern blot analysis using probes for Fos proto-oncogene, D14S20 marker type IV collagenase gene, haptoglobin gene located on chromosome bands 14q and 16q did not show any loss or rearrangement occurred in these subclones. Similarly, no loss or rearrangement was observed for the krev-1 gene on chromosome 1, whose number was not changed in either subclone. These findings indicate that in CI- 1, CIII, and CVII cells, the number of chromosome band 10q is decreased relative to HT29 cells. These deletions may be implicated in the morphological differentiation of HT29 cells treated with sodium butyrate.

Expression and regulation of the 67-kda laminin-binding protein and its precursor gene in lymphoid-cells.

The 67-kDa laminin-binding protein is a non-integrin laminin-binding protein that mediates cancer cell adhesion and migration. The expression of the 67-kDa laminin-binding protein and of its putative precursor, a 37-kDa polypeptide, was studied in peripheral T-cells and T-lymphoma cell lines. Immunofluorescence experiments detected antigen in both the cytosol and on the cell membrane. On immunoblots of T-cell protein extracts, both the 37-kDa precursor and the mature 67-kDa protein were present. The mRNA for the precursor was expressed in both immature and mature thymocytes. In three independent T-lymphoma cell lines, the mRNA levels were decreased after prolonged stimulation with phorbol esters. Since the latter directly activate protein kinase C, it appears that regulation of the 37-kDa precursor in T-cells may be mediated by the signal transduction cascade associated with protein kinase C activation.

Genetic and epigenetic events in human hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is the most frequently occurring liver carcinoma world-wide. Clinical and molecular medical analyses have produced a considerable amount of information about liver carcinogenesis. Loss of heterozygosity (LOH) analyses have revealed several chromosomal loci harboring potential tumor suppressors. These data support the idea that deletion or inactivation of tumor suppressors including RB, p53, BRCA2, E-cadherin and other candidate genes seem to be common events in HCC development. Factors associated with cell cycle regulation via the Wnt- and MAPK/ERK signaling pathways are frequently deregulated in hepatocarcinogenesis. Aberrant activation of telomerase also occurs in precancerous as well as cancerous lesions in HCC patients. To characterize the wide variety of genetic events that occur in HCC, mRNA expression has been compared in HCC and non-cancerous liver tissues, and several differentially expressed genes have been identified. Hepatitis B and C viruses are the main risk factors for HCC, and indeed some accessory functions of viral products seem to contribute to tumor development; however, whether they have a direct carcinogenic effect has not yet been established.

Extraadrenal expression of steroid 21-hydroxylase and 11 beta-hydroxylase by a benign testicular Leydig cell tumor.

We present an unusual case with bilateral testicular Leydig cell tumors displaying extraadrenal expression of steroid 21-hydroxylase and 11 beta-hydroxylase. Histological examination of a 38-yr-old man infertile due to azoospermia showed him to have bilateral testicular Leydig cell tumors. The in vitro steroidogenic potential of the tumors and their adjacent testicular tissue was evaluated using organ culture. Tumor tissue was found to secrete deoxycorticosterone (DOC), corticosterone (B) and cortisol, which are not produced in normal adult testis, into the medium, while testicular tissue adjacent to the tumors secreted a small amount of DOC and B. Northern blot analysis with cytochrome P-450C21 complementary DNA (cDNA) and P-45011 beta cDNA as probes revealed that the tumor contained a considerable amount of mRNA for P-450C21 and P-45011 beta, while the MRNAs were not detected in the testicular tissues adjacent to the tumors. It is suggested that the high local levels of estrogen and/or progesterone within the Leydig cell tumors and their adjacent testicular tissues induced extraadrenal expression of steroid 21-hydroxylase and 11 beta-hydroxylase by the tumors and their adjacent testicular tissues.

Enhanced expression of S8, L12, L23a, L27 and L30 ribosomal protein mRNAs in human hepatocellular carcinoma.

Differential display (DD) analysis using surgically resected human hepatocellular carcinoma (HCC) and adjacent non-tumorous liver tissues was performed. We identified 5 cDNAs up-regulated in human hepatocellular carcinoma, encoding S8, L12, L23a, L27 and L30 ribosomal protein mRNAs. Northern blot analysis, using total RNAs from thirteen pairs of HCC and abjacent non-tumorous liver tissues demonstrated that these mRNA levels were up-regulated along with the histological grading of tumors. The expression of these mRNAs was also high in three human HCC cell lines (HuH-7, HepG2 and HLF), irrespective of the growth state. These results suggest that activation of these genes is an important manifestation of HCC phenotypes.

Enhanced expression of translation factor mRNAs in hepatocellular carcinoma.

Several studies have demonstrated elevated expression of translation factor mRNAs in malignant tissues. In this study, using primary human hepatocellular carcinoma (HCC) tissues, we examined gene expression of translation factors, including 2 eukaryotic initiation factors (eIFs-4A1, -4E), 4 elongation factors (eEFs-1 alpha, -1 gamma, -1 delta, and -2) and 10 ribosomal proteins (Rps P1, P2, S10, L35, L5, L39, L9, L6, S3a and S17), whose mRNA expression has never been examined in HCC. Our results demonstrated that all the mRNAs examined were up-regulated in HCC tissues. Among 7 HCC tissues of different histological grades, the expression of these mRNAs remained at basal levels in a well to moderately differentiated (W/M-) HCC, was coordinately up-regulated in moderately differentiated (M-) HCCs. In moderately to poorly differentiated (M/P-) HCCs, the expression of eEFs-1 gamma, -1 delta, -2, Rps P0 and L9 mRNAs was further up-regulated along with the histological grading. These results therefore suggest that coordination and specific activation of translation factor genes might be involved in the process of liver carcinogenesis.

Neurocysticercosis without detectable specific antibody.

A 19-year-old girl who had lived in India for five years until 1992 was admitted to Hokuto Hospital after general seizures which lasted for fifteen minutes. Cerebral magnetic resonance imaging (MRI) showed a ring-enhanced lesion of 6 mm in diameter in the right parietal lobe. She underwent surgical resection after diagnosis of the brain tumor. Histopathological examinations revealed that the resected tumor was a cysticercus of Taenia solium (T. solium), and we concluded that her seizures were caused by neurocysticercosis. Serological examinations by enzyme-linked immunosorbent assay (ELISA) and immunoblots to detect specific antibody against the glycoproteins of T. solium showed no detectable antibody response. The patient is under careful observation in our out-patient clinic with no medication.

Single solitary metastasis of the slowly progressive type of renal cell carcinoma to the choroid plexus--case report.

A 68-year-old male developed a solitary metastasis in the choroid plexus of the right lateral ventricle 7 years after left nephrectomy for renal cell carcinoma. The lesion was totally removed and the patient has been free from recurrence for 30 months postoperatively. Pure solitary metastasis to the choroid plexus is very rare, but five of the 15 cases originated from renal cell carcinoma. There may be a link between this type of metastasis and the slowly progressive type of renal cell carcinoma.

Correlative changes of plasma renin activity and serum testosterone in human internal spermatic vein after single administration of human chorionic gonadotropin.

To study changes in renin secretion from the human testis at various days after hCG treatment, plasma renin activity in the internal spermatic vein (ISV) and cubital vein (CV) was measured at the time of surgical repair of varicocele in 79 patients. Sixty-five of them were given a single administration of hCG (10,000 IU/m2) 1 to 10 days before operation. Although the mean PRA levels in CV in both treated and untreated groups were similar, the mean PRA levels in ISV obtained 1 to 5 days after hCG treatment were significantly higher than that in the untreated group. However, the mean PRA levels in ISV returned to that in the untreated group 6-10 days after treatment. Serum testosterone levels in the same ISV were also much higher 1 to 6 days after hCG treatment, compared with those in the untreated group. On the basis of these results it is concluded that (a) the secretion of renin from the testis into ISV can be demonstrated for 5 days after hCG treatment; (b) the parallel increases in PRA with testosterone in ISV seem to suggest a possible influence of androgen on renin production in Leydig cells of human testis.

[Clinical results of transrectal hyperthermia in 15 patients with chronic abacterial prostatitis].

A prospective uncontrolled study of the safety and efficacy of transrectal hyperthermia was performed on 15 patients with chronic abacterial prostatitis or prostatodynia. A total of 6 (1-2 per week) 1-hour sessions of hyperthermia were performed. Subjective improvement was fair in 2 cases and slight in 13 cases (about 87%). The uroflowmetry and the prostatic size measured by transrectal echography did not show any significant changes. The complications were epididymitis (1 case), UTI (1 case) and chlamydial urethritis (2 cases). In conclusion, this therapy seems to achieve the short-term improvement of chronic abacterial prostatitis or prostatodynia.