Expression of manganese superoxide dismutase in esophageal and gastric cancers.

The tumor-killing activity of radiotherapy and chemotherapy for cancer is closely associated with the production of active oxygen, and the relation between therapeutic resistance and active oxygen scavengers produced by the tumor itself is gaining more attention. It is considered that manganese superoxide dismutase (MnSOD) protects host cells from oxidative stress, in synergy with other antioxidant enzymes. In this study, we used a quantitative polymerase chain reaction assay to measure MnSOD mRNA in resected specimens from patients with esophageal and gastric cancers. In both esophageal and gastric cancers, the level of MnSOD mRNA was significantly elevated in cancer tissue compared to non-cancer tissue (P < 0.01). In gastric cancer tissue, the MnSOD mRNA level was significantly higher than in esophageal cancer tissue (P < 0.01). The significance of MnSOD in cancer tissue was investigated further by measuring MnSOD content in resected specimens using an enzyme-linked immunosorbent assay, and by examining its location by an immunohistochemical method. Upregulation of MnSOD in cancer tissue most likely serves as a protective mechanism against anti-cancer therapies known to produce superoxide radicals as a key component of their tumor-killing activity.

Enhanced expression of manganese superoxide dismutase mRNA and increased TNFalpha mRNA expression by gastric mucosa in gastric cancer.

Manganese superoxide dismutase (Mn-SOD), a mitochondrial enzyme, is a cytokine-regulated acute-phase protein that protects cells from free radicals. The current investigations examined the in vivo regulation of the expression of Mn-SOD mRNA and tumor necrosis factor alpha (TNFalpha) mRNA in gastric carcinoma tissue. The expression of these transcripts in breast carcinoma tissue also was examined because breast cancer is a much more TNF-sensitive tumor than gastric cancer. TNFalpha mRNA was markedly increased in gastric carcinoma tissue (p < 0.005). There were significantly higher levels of Mn-SOD mRNA in gastric carcinoma tissue than in noncancerous tissue (p < 0.0001). The level of Mn-SOD mRNA in gastric carcinoma tissue was higher than that in breast carcinoma tissue (p < 0.005). Up-regulation of Mn-SOD mRNA in gastric carcinoma tissue most likely serves as a protective mechanism against superoxide radicals and TNF cytotoxicity.

RANTES mRNA expression in skin and colon of patients with atopic dermatitis.

The expression of RANTES mRNA in dermal and colonic tissue was examined in patients with atopic dermatitis by the reverse transcription polymerase chain reaction method. RANTES mRNA was detected in the colon in 8 of 10 patients and in 1 of 5 control patients. It was present in rashes in 9 of 10 patients and at non-eruptive sites in 5 of 7 patients. These findings suggest that RANTES is involved in eosinophil infiltration and T cell infiltration in atopic dermatitis.

Induction of gene expression for immunomodulating cytokines in peripheral blood mononuclear cells in response to orally administered PSK, an immunomodulating protein-bound polysaccharide.

The protein-bound polysaccharide extracted from a fungus, PSK, has been used as a biological response modifier in the treatment of cancer patients in Japan for over 16 years. The administration of PSK to tumor-bearing rodents inhibited tumor growth and modulated immune responses. Recently, an in vitro study has revealed that PSK is a strong inducer of cytokine gene expression and production in human peripheral blood mononuclear cells (PBMC). To establish whether PSK has cytokine-inducing activities in vivo, we have orally administered PSK (1 g, the clinical dose) to 12 healthy volunteers and 9 gastric cancer patients who had undergone gastrectomy, and assessed the gene expression for cytokines in PBMC of each subject. As determined by the reverse-transcribed polymerase chain reaction method, the induction of gene expression for both tumor necrosis factor alpha and interleukin-8 (IL-8) was detected in PBMC from 5 of the 12 healthy volunteers (42%) and 4 of the 9 patients (44%). Furthermore, the concentration of serum IL-8 was elevated in 5 healthy volunteers given PSK orally, who had shown induction of IL-8 gene expression, as detected by enzyme-linked immunosorbent assay. These findings indicate that responsiveness of PBMC to PSK, in terms of gene expression and production of cytokines, varies among individuals. Thus, when using PSK to treat cancer patients, it seems advisable to select patients on the basis of their responsiveness to PSK. We speculate that the cytokines induced by PSK might mediate the immunoenhancing action of this agent in vivo.

Increased expression of interleukin-8 mRNA in ulcerative colitis and Crohn's disease mucosa and epithelial cells.

: Interleukin-8 (IL-8) is a chemotactic cytokine (chemokine), which both attracts and activates granulocytes. IL-8 could have a central function in the initiation and perpetuation of the inflammatory bowel diseases (IBD), due to its relative resistance to inactivation and long half-life in vivo. Using a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay, we have observed elevated levels of IL-8 mRNA in colonic mucosal sections obtained from surgically resected specimens from ulcerative colitis (UC) and Crohn's disease (CD) patients with actively inflamed mucosa. The level of IL-8 mRNA expression in the intestinal mucosal biopsies from UC and CD patients was much greater in involved as opposed to noninvolved mucosal sections. The highest expression of IL-8 mRNA detected by RT-PCR was in UC mucosa and in isolated intestinal epithelial cells from UC patients. Increased IL-8 production by cells in IBD intestinal mucosa as well as IBD epithelial cells may be involved in the continuous attraction and activation of granulocytes in the inflamed intestine in both UC and CD patients. Chemokines, such as IL-8, are potent chemoattractant molecules and may have a central role in the augmentation and perpetuation of inflammation in IBD.

Quantitative PCR for detection of femtogram quantities of interleukin-8 mRNA expression.

A quantitative polymerase chain reaction (PCR) assay for mRNA expression of interleukin-8 (IL-8), a neutrophil chemotactant and activator, was developed to examine the expression of this cytokine by colonic mucosa. A synthetic IL-8 RNA deleted in size of native IL-8 mRNA was used as an external control. The synthetic IL-8 RNA was mixed with total RNA from cells and converted to cDNA and amplified by PCR simultaneously. The lower limit of sensitivity for the assay was found to be more than 1 femtogram of IL-8 mRNA. The assay determined IL-8 mRNA expression when the RNA was isolated from either human histiocytic lymphoma cell line U937 cells or human colonic mucosa obtained from colitis patients and healthy controls. The development of the rapid and sensitive assay should provide a means to more fully evaluate the role of this cytokine in diverse disease states with small scale.

Infection of terminally differentiated myotubes with Rous sarcoma virus (RSV): lack of DNA integration but presence of RSV mRNA.

We re-examined the generally accepted concept that replication of Rous sarcoma virus (RSV) requires host DNA synthesis. We used terminally differentiated chicken myotubes as the host because chromosomal DNA replication is completely abolished by the natural differentiation process. Southern blot analysis detected unintegrated viral DNA in both the nucleus and cytoplasm of infected myotubes. This indicated that reverse transcription of the infecting viral RNA and transport of the newly synthesized viral DNA into the nucleus proceeded normally in myotubes. However, restriction enzyme digestion of high M(r) DNA prepared from infected myotubes produced none of the fragments specific for RSV, indicating that the viral DNA had failed to integrate into the myotube chromosomal DNA. In these infected myotubes, viral RNA was detected by in situ hybridization. Northern blot analysis showed the presence of all three RSV mRNAs (38S, 28S and 21S). The amount of these viral RNAs in infected myotubes was comparable with that found in infected fibroblasts. We conclude that host DNA synthesis is required for RSV integration, but, in contrast to the generally accepted concept, viral DNA integration is not an absolute requirement for transcription of the RSV genome.

Infection of terminally differentiated myotubes with Rous sarcoma virus: reduced synthesis of env and v-src proteins.

We studied Rous sarcoma virus (RSV) protein synthesis in RSV-infected, terminally differentiated chicken myotubes ('late-infected' myotubes), in which no viral DNA integration takes place but all three viral mRNAs (38S, 28S and 21S) are transcribed normally. With the use of specific anti-RSV protein antisera, we found that only the viral gag and pol proteins were synthesized at levels similar to those synthesized in RSV-transformed fibroblasts; the synthesis of env and v-src proteins was significantly reduced in these infected myotubes. We concluded that the viral RNA transcribed from the unintegrated RSV DNA was functional but that genes at the 3' end of the RSV genome were translated at a lower level. By contrast, when mononucleated replicating chicken myoblasts were infected with a mutant (tsNY68) carrying a temperature-sensitive v-src gene and maintained at the non-permissive temperature for this gene, they developed into myotubes with viral DNA integrated in their chromosomal DNA. These 'early-infected' myotubes expressed all four viral genes (gag, pol, env and v-src) at a level similar to that in infected fibroblasts. This result ruled out the possible presence of specific factor(s) in myotubes that preferentially inhibit the 3' genes of RSV, and suggested other translational control(s) of viral gene expression in late-infected myotubes.

[Study of in vitro OK432-induced IFN-gamma production in patients with gastric and esophageal cancers: comparison with in vitro skin tests].

In the present study, we measured in vitro production of IFN-gamma induced by OK432 in peripheral mononuclear cells (PMC) and investigated the relationship between the skin reaction to Su-Ps, an extract of Streptococcus Pyogenes, and purified protein derivative (PPD) and the productibility of IFN-gamma in patients with gastric and esophageal cancers. PMC isolated with the Ficoll-conray centrifugation technique were adjusted to 10(6) cells/ml in RPMI-1640 medium supplemented with 10% fetal calf serum and incubated over 7 days at 37 degrees C in the presence of OK432 at 0.17 KE/ml in microculture plates. IFN-gamma secreted in the supernatants was measured consecutively during the observation period with radioimmunoassay. The results are summarized as follows. 1. The production of IFN-gamma in PMC from patients with negative skin reaction to Su-Ps and PPD was significantly decreased as compared with the value obtained from patients who were positive in these skin tests (p less than 0.001). 2. In vitro production of IFN-gamma induced by OK432 was significantly correlated with the degree of skin tests to Su-Ps and PPD as well (r = 0.48, r = 0.41, p less than 0.01, respectively). Thus, it is concluded that the assay of IFN-gamma produced in PMC cultures is useful to evaluate the immunological status of patients with cancer.

[OK 432-induced production of IFN-gamma in peripheral mononuclear cells from healthy subjects and patients with cancer].

OK 432, a well-established immunopotentiator, has been recently noticed as one of biological response modifiers (BRM). IFN-gamma, also called immune interferon, is regarded as an important immunoregulator secreted by T-lymphocytes. In the present study, we measured the in vitro production of IFN-gamma in human peripheral mononuclear cells (PMC) induced by OK 432. PMC were isolated from the peripheral blood with the Ficoll-Conray centrifugation technique. The number of cells for culture was adjusted to 1 x 10(6) cells/ml in RPMI-1640 medium supplemented with 10% fetal calf serum. Incubation was performed over 7 days at 37 degrees C in the presence of OK 432 at 0.17KE/ml in microculture plates. IFN-gamma secreted in the supernatants was measured consecutively during the observation period with radioimmunoassay. IFN-gamma production in PMC from healthy subjects was already detectable at 24 hours of culture and elevated gradually with incubation, reaching as much as 94.3 +/- 44.6 u/ml (mean +/- SD) after 7 days of culture. In contrast, the production of IFN-gamma in patients with cancer was severely suppressed as 21.9 +/- 25.4 u/ml after 7 days of culture (p less than 0.01). Furthermore, both surgical and radiation treatments inhibited the production of IFN-gamma in PMC from patients with cancer.