Akio Hizuta

The p53 gene is a potent determinant of chemosensitivity and radiosensitivity in gastric and colorectal cancers

We previously reported that introduction of the wild-typep53 gene into human cancer cells with deletedp53 enhanced apoptosis induced by chemotherapy [Fujiwara et al. (1994) Cancer Res 54∶2287]. This suggests thatp53 status could be a potent determinant of the therapeutic efficacy of DNA-damaging cancer therapy. We analyzed 24 patients with gastric or colorectal cancer forp53 mutations and apoptotic changes in surgical specimens. Out of 11 patients with gastric cancer, 3 were treated with chemotherapeutic drugs before resection; 5 of 13 patients with colorectal cancer had 30 Gy radiation prior to surgery.p53 mutations were detected in 4 cases of gastric cancer (36.4%) and in 6 cases of colorectal cancer (46.2%) by immunohistochemical staining. The preoperative DNA-damaging therapies increased the number of apoptotic cells in wild-type-p53-expressing tumors; tumors with mutantp53, however, significantly showed fewer apoptotic cells compared with those expressing wild-typep53. Thep53-inducible WAF1/CIP1 protein was immunohistochemically observed in wild-type-p53-containing tumors, where-as mutant-p53-expressing tumors expressed no detectable WAF1/CIP1. Taken together, we conclude thatp53 mutations are associated with the poor response of chemotherapy and radiotherapy.

p53 expression overcomes p21WAF1/CIP1-mediated G1 arrest and induces apoptosis in human cancer cells.

The p21WAF1/CIP1 gene, which encodes a cyclin-dependent kinase inhibitor, may be critical for tumor suppressor gene p53-induced cell cycle arrest. The p53 gene is known to regulate G1 checkpoint, which can either induce G1 arrest or initiate apoptosis. To directly examine the role of p21WAF1/CIP1 in the control of p53 function, we have introduced human p21WAF1/CIP1 gene into a p53-deficient human non-small cell lung cancer cell line H1299 using a p21WAF1/CIP1-expressing adenoviral vector (AdCMVp21). Infection with AdCMVp21 resulted in high levels of p21WAF1/CIP1 expression and significantly suppressed the growth of H1299 cells through the G1 arrest of the cell cycle. In contrast, transient expression of the wild-type p53 gene by a recombinant adenoviral vector (AdCMVp53) in H1299 cells induced apoptotic cell death and resulted in a rapid loss of cell viability. We then examined the effects of combined infection with AdCMVp21 and AdCMVp53 on H1299 cells to explore the dominant function of these molecules. Interestingly, introduction of exogenous p53 overcame p21WAF1/CIP1-mediated cell cycle arrest at G1 and induced apoptosis, although viral-transduced p21WAF1/CIP1 expression level was unaffected. These observations suggest that p53 expression converts a p21WAF1/CIP1-mediated growth arrest into apoptosis. The result was repeated with two additional human colon adenocarcinoma cell lines with the different p53 status, mutant p53-expressing DLD-1 and wild-type p53-expressing LoVo, suggesting that this phemonenon is a general event among human cancer cells. Thus, p53-mediated apoptotic pathway is dominant over the growth arrest pathway, indicating that p53 may be an essential upstream mediator of p21WAF1/CIP1 in the regulation of a cell process leading either to growth arrest or to apoptotic suicide.

Differential involvement of the CD95 (Fas/APO-1) receptor/ligand system on apoptosis induced by the wild-type p53 gene transfer in human cancer cells

The CD95 (Fas/APO-1) system regulates a number of physiological and pathological processes of cell death. The ligand for CD95 induces apoptosis in sensitive target cells by interacting with a transmembrane cell surface CD95 receptor. We previously reported that the recombinant adenovirus-mediated transfer of the wild-type p53 gene caused apoptotic cell death in a variety of human cancer cells. To better understand the mechanism responsible for this cell death signaling, we have investigated the potential involvement of the CD95 receptor/ligand system in p53-mediated apoptosis. The transient expression of the wild-type p53 gene up-regulated the CD95 ligand mRNA as well as protein expression in H1299 human lung cancer cells deficient for p53 and in DLD-1 and SW620 human colon cancer cells with mutated p53, all of which constitutively expressed CD95 receptor as shown by a flow cytometric analysis, and induced rapid apoptotic cell death as early as 24 h after gene transfer. However, the sensitivity to the cytolytic effect of agonistic anti-CD95 antibody (CH11) varied among these cell lines: CH11 induced apoptosis in H1299 cells, but not in DLD-1 and SW620 cells despite their abundant CD95 receptor expression, suggesting that the CD95 receptors on DLD-1 and SW620 cells might be inactivated. In addition, an antagonistic anti-CD95 ligand antibody (4H9) that interfered with the CD95-receptor-ligand interaction partially reduced the apoptosis induced by the wild-type p53 gene transfer in H1299 cells, whereas apoptosis of DLD-1 and SW620 cells occurred in the presence of 4H9. Taken together, these findings led us to conclude that the CD95 receptor/ligand system is differentially involved in p53-mediated apoptosis, suggesting that the restoration of the wild-type p53 function may mediate apoptosis through CD95 receptor/ligand interactions as well as an alternative pathway.

Novel combination therapy for human colon cancer with adenovirus- mediated wild-type p53 gene transfer and DNA-damaging chemotherapeutic agent

Alteration of the wild‐type (wt) p53gene by mutation, deletion or re‐arrangement is a major factor in the development of human colon cancer. Recent studies have demonstrated that p53 might be an essential component of the apoptotic pathway triggered by DNA‐damaging stimuli such as chemotherapeutic agents and ionizing radiation. We examined the anti‐tumor effects of adenovirus‐mediated wt‐p53 gene transfer in combination with a chemotherapeutic drug on the human colon cancer cell line WiDr, which is homozygous for a mutation in the p53 gene. Treatment with the chemotherapeutic drug cisplatin following infection with a replication‐deficient, recombinant adenoviral vector expressing wt‐p53 (termed AdCMVp53) significantly suppressed the growth of WiDr cells compared to single treatments alone. To evaluate the in vivo efficacy of AdCMVp53 and cisplatin given sequentially, WiDr cells were inoculated s.c. in nu/nu mice. After 3 days, AdCMVp53 was injected s.c. into the area where tumor cells were implanted, followed by i.p. administration of cisplatin. Analysis of initial growth inhibition at 21 days demonstrated a profound therapeutic cooperativity, though administration of either AdCMVp53 or cisplatin alone was followed only by a slowing of growth. Our results suggest that gene therapy using wt‐p53‐expressing adenovirus in combination with a chemotherapeutic DNA‐damaging drug could be a useful strategy for treating human colon cancer. Int. J. Cancer 73:367–370, 1997.

Localization of T cell receptor (TCR)- γδ+ T cells into human colorectal cancer: flow cytometric analysis of TCR-γδ expression in tumour-infiltrating lymphocytes

We analysed TCR‐γδ expression in tumour‐infiltrating lymphocytes (TIL) obtained from 13 patients with colorectal cancer and simultaneously isolated the T lymphocytes from normal intestinal tissue (IL) to compare the frequencies of TCR‐γδ expression in TIL, IL. and peripheral blood lymphocytes (PBL) in the same patient. Flow cytometric analysis showed that the frequency of TCR‐γδ expression in TIL (275 ± 1·84%) was significantly lower than that in IL (15·28 ± 9·45%, P < 001). However, a larger quantity of TIL was separated than IL per unit weight of specimen, so the total number of γδ T cells obtained per unit weight was not different between tumour tissue and normal intestine. In addition, phenotypic analysis revealed that about half of the TCR‐γδ TIL were CD8+ (CD4+, 3·0 ± 3·1%; CD8+, 54·7 ± 19·9%, mean ± s.d. of five patients), and a very similar result was obtained in TCR‐γδ+ IL (CD4+ 2·7 ± 2·4%; CD8+, 53·1 ± 17·4%). In contrast, most TCR‐γδ+ PBL were double‐negative (CD4+. 3·2 ± 3·0%; CD8+ 20·6 ±7·4%). These results indicated that TCR‐γδ+ CD8+ T cells selectively and consistently localized in colorectal tumour tissue, similarly to normal intestinal epithelium.

Decreased Serum Tryptophan in Patients with Cancer Cachexia Correlates with Increased Serum Neopterin

We investigated serum tryptophan (Trp), neopterin (NPT) and immunosuppressive acidic protein (IAP), one of tumor-associated tumor marker, concentrations in 28 patients with gastrointestinal tumors representing cancer cachexia and 10 healthy controls. NPT comes from activated macrophages presumably activated by tumor-sensitized T cells via gamma-interferon (IFN-gamma) excitation of the macrophages. We found that the NPT level was significantly higher than the control value. The negative correlation of NPT and Trp concentrations indicates activity of indoleamine 2,3-dioxygenase (IDO), a Trp degradating enzyme, in cancer-burden patients. The activity of IDO can be induced by cytokines such as IFN-gamma, and therefore low Trp levels may result from endogenous IFN-gamma production due to immune activation against tumors. We also found a positive correlation between NPT and IAP levels, suggesting that host immune activation against tumors played a role in the immunosuppression of cancer-burden states, followed by cancer-cachexia.

Appendiceal mucocele with concomitant colonic cancer : report of two cases

PURPOSE: Mucocele of the appendix is an uncommon disorder, usually found incidentally during ultrasonography or radiographic studies. We report two cases of combined appendiceal mucocele and colonic cancer. METHODS: The two cases were analyzed for the clinicopathologic characteristics such as history, presentation, laboratory data, radiologic and endoscopic studies, pathology, and p53 immunoreactivity. RESULTS: Two patients were diagnosed with an appendiceal mucocele by ultrasound of the abdomen, together with computed tomography. Colonoscopic examination subsequently revealed synchronous colonic adenocarcinoma in both patients. Ileocecal resection following endoscopic polypectomy and a right hemicolectomy was performed for each patient. An appendiceal mucocele was histologically diagnosed as a mucinous cystadenoma. Immunohistochemical detection of abnormally high level of p53 protein was observed in colonic adenocarcinomas of both patients, whereas both appendiceal cystadenomas were negative for p53. CONCLUSIONS: To be remembered is the high frequency of concomitant gastrointestinal tumors in patients with appendiceal mucocele, especially caused by mucinous neoplasms. A total colonoscopic surveillance will afford earlier diagnosis of synchronous colonic cancers in these patients.

Functional expression of Fas and Fas ligand on human intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IEL) constitute the first lymphoid compartment to encounter dietary antigens and intestinal pathogens. IEL are proposed to be involved in the defence against bacterial and viral invasion and to play an important role in mucosal immunity. Fas (CD95/APO‐1) is a surface receptor that induces apoptotic cell death upon ligation with Fas ligand (FasL). The aim of this study was to examine the expression and function of Fas and FasL on freshly isolated normal human colonic IEL. The expression and function of Fas and FasL on IEL isolated from 40 normal colonic specimens were examined by flow cytometry, reverse transcriptase‐polymerase chain reaction, immunohistochemistry, and DNA‐release cytotoxicity assay. Virtually all CD3+ IEL (95.2 ± 4.3%) expressed Fas and were sensitive to agonistic anti‐Fas antibody, whereas only 56.6 ± 8.4% of peripheral T lymphocytes expressed Fas and were resistant to the antibody. We also detected FasL mRNA and protein (40.1 ± 4.2%) on IEL, and found that IEL exerted FasL‐mediated cytotoxicity against Fas‐expressing target cells. These findings suggest that human IEL are activated in situ but are tightly regulated by the constitutive expression of functional Fas and FasL to maintain homeostasis of the mucosal immune system.

Interleukin-1 Receptor Antagonists and Other Markers in Colorectal Cancer Patients

BACKGROUND Although the interleukin-1 receptor antagonist (IL-1ra) has been suggested as a potentially valuable therapeutic agent and has been shown to improve outcome in various animal models of arthritis, septic shock, and inflammatory bowel disease, there is little information available about its level in the circulation in patients with cancer. METHODS Serum levels of IL-1ra, soluble interleukin-2 receptor (sIL-2r), soluble intercellular adhesion molecule-1 (sICAM-1), and cortisol were measured in normal controls and patients with colorectal cancer. RESULTS The data showed that serum IL-1ra levels in patients were significantly lower than those of healthy controls (P < 0.05). In contrast, serum sIL-2r and cortisol levels in patients were significantly higher than those of normal controls (P < 0.01). Serum sICAM-1 levels in patients were the same as in normal controls. CONCLUSIONS These results suggested that a reduced level of IL-1ra exists in colorectal cancer patients relative to normal controls, indicating that cancer patients have an immunologic disorder and that exogenous IL-1ra administration might be a future alternative for cancer treatment.

Enhancement of antitumor cytotoxicity of hepatic lymphocytes by oral administration of PSK

We have studied the effects of oral administration of a biological response modifier (BRM), PSK, on hepatic lymphocytes. Many PSK positive cells were observed in the liver by anti-PSK antibody staining. Flow cytometric analysis revealed an increase in the number of OX8 (CD8) positive cells in the non-parenchymal nonadherent liver cells (NPNALCs) which were isolated from the liver enzymatically digested by perfusion with collagenase. NPNALCs were fractionated by discontinuous density gradient centrifugation, and the number and cytotoxic activity of these cells were examined in each fraction. Although the yield of lymphocytes in each fraction was not significantly increased by the oral administration of PSK, the natural killer (NK) activity was markedly enhanced in low density fractions. The present findings suggest that oral administration of PSK is effective for prevention of liver metastasis through the augmentation of organ-associated NK activity.