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We recently reported that three members of the integrin family of cell adhesion molecules, designated alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4, are expressed at increased levels within the tumors and cell lines of patients with SCC. These three integrins have been reported to serve as receptors for laminin isoforms, and we also previously observed that laminins are secreted by SCC cell lines isolated from patients. In this study, the expression and localization of the three integrins and laminin in situ was evaluated in ten tumor specimens from patients with SCC by immunohistochemistry using integrin subunit-specific monoclonal antibodies. The ability of the antibodies to inhibit laminin attachment of a human squamous cell carcinoma line was determined by in vitro cell adhesion assay. Laminin and the three integrins were co-localized along the invasive border of the tumor parenchyma in 10/10 patient tumor specimens. Attachment of the UM-SCC-38 cell line to laminin was strongly inhibited by specific mAbs to alpha 2 and alpha 6 integrin subunits alone, or completely using a combination of alpha 2, alpha 3, and alpha 6 subunit specific mAbs. The co-localization of the three abnormally expressed integrins and laminin in patient tumor specimens indicates the potential for interaction of these receptors and ligand in vivo. The results of the cell adhesion assays using a patient SCC cell line that expresses the same repertoire of integrins confirms that SCC attach to laminin isoforms primarily through the alpha 2, alpha 3 and alpha 6 subunit-containing integrins. These findings provide a basis for undertaking experimental studies to obtain small molecule receptor antagonists to determine the role of these integrins in tumor formation, growth, invasion and metastasis in vivo.

beta-catenin is a multifunctional protein: it plays a central role in the cell-cell adhesive junctions, and participates in transduction of the morphogenic Wingless/Wnt-signal. Upon detailed analysis of the human beta-catenin gene, an intragenic polymorphic microsatellite marker could be identified. This marker shows 62% heterozygosity and was used in a study of eleven different tumor types. A high level of beta-catenin allelic imbalance was observed for small cell lung carcinoma, squamous cell lung carcinoma and cervix carcinoma. Other microsatellite markers on 3p24-21 could demonstrate frequent but not invariable codeletion of flanking chromosomal loci. This intragenic polymorphic marker will allow selection of tumor types and tumor samples possibly bearing recessive mutations in the remaining allele of the beta-catenin gene.

Human chronic myelogenous leukemia (CML) is a unique malignancy in its cellular and molecular phenotypes. High dose therapy followed by stem cell transplantation seems to be one of the most effective treatment modalities for CML. However, allogeneic stem cell transplantation, a curative treatment modality, is limited due to the availability of matched siblings. On the other hand, the autologous stem cell harvests are contaminated with leukemic cells, and therefore a significant reduction of leukemic cells is desired before using the harvest for transplantation. Therefore in the present study, effects of a combination of a suboptimal concentration of 4-hydroxyperoxycyclophosphamide (4HC) and an optimal concentration of c-myb antisense oligonucleotide on the growth of K562 human chronic myelogenous leukemic cells in vitro were determined. The combination significantly (p<0.05) inhibited the growth of K562 cells in vitro when compared to the effects of c-myb oligonucleotide or 4HC alone. The c-myb oligonucleotide alone or in combination with low dose 4HC decreased the expression of c-myb gene as determined by RT-PCR techniques. Cellular uptake and retention of fluoresceinated oligonucleotide in control and treated K562 cells was studied using plain field laser microscopy and flow cytometry. There was an increase in cellular uptake of c-myb oligonucleotide in K562 cells as measured by plain field laser microscopy in the presence of 4HC. The combination of oligonucleotides and 4HC did not significantly decrease the number of hematopoietic stem/progenitor cells from normal hematopoietic stem cell harvests as determined by in vitro colony assays. The combination of low dose 4HC and c-myb antisense oligonucleotides can potentially be applied in CML patients, particularly for purging leukemic cells present in their hematopoietic stem cell harvests.

In most normal somatic cells the telomeres of human chromosomes shorten with each cell division because of low expression or lack of telomerase activity. Telomerase, a ribonucleoprotein that synthesizes telomeric DNA onto chromosomal ends, is reactivated or upregulated in tumor cells and maintains the stability of telomere length. We previously showed that treatment of HL60 leukemia cells with differentiation-inducing agents resulted in inhibition of telomerase activity. In the present study, we found that the decrease in telomerase activity did not temporally correlate with the expression of a differentiation marker, CD11b, on the cell surface. Mixing of protein extracts from telomerase-negative differentiated HL60 cells with those from parental HL60 cells did not result in inhibition of telomerase activity, suggesting that a diffusible cellular telomerase inhibitor was not produced in the differentiated cells. However, a decrease in telomerase activity correlated with a selective decrease of telomerase RNA expression, a decrease in the levels of total cellular RNA, and an increase in cells at G(0) phase.

Length of terminal restriction fragments (TRFs) was examined in 34 samples from hepatocellular carcinoma (HCC) patients. TRF length alterations (reduction or elongation) were found in 18 of 34 (53%) HCC nodules (13 cases reduced, 5 cases elongated). The incidence of TRF alteration was significantly higher in HCC exceeding 3 cm in diameter, moderately- or poorly-differentiated in histology, or with microscopic aggressive lesions (P<0.05). The survival rates were significantly higher in patients without alterations in TRFs (P<0.05). Specifically, all 5 cases with elongated TRFs recurred within 1 year after resection and had a poor prognosis. In contrast, the presence of telomerase activity was not associated with alterations in TRF length. The incidence of TRF length alterations increased with HCC tumor progression.

Loss of chromosome sequences at 13q14 (Rbl) and 17p13 (p53) associated with squamous cell carcinoma of head and neck (SCCHN) was evaluated in 12 recurrent tumors and 51 primary tumors from 63 patients. The incidence of LOH at 17p13 was 19 of 50 (38%) tumors, and at 13q14 was 21 of 57 (37%). LOH affecting Rbl and/or p53 was observed in 30 of 63 (48%) SCCHN. Coincident LOH at Rbl and p53 was detected in 10 of 46 (22%) tumors. There were nine cases in which primary and metastatic tumors were obtained from the same patient. Of these, seven were informative and five of these (71%) manifested LOH at p53 in both primary and metastatic sites. Examination of Rbl in these same tumors showed LOH in six of the nine metastases, and of these six, only three revealed LOH in the primary tumor. LOH at p53 or Rbl alone showed no correlation with clinical outcome. However, tumors that manifested LOH at both loci were associated with poorer patient outcome and poorer histological differentiation.

Certain chymotrypsin-like protease inhibitors such as TPCK exhibit a well described anti-tumorigenic activity by an as yet undescribed mechanism. One potential cellular target for TPCK in transformed cells is the ms-inducible NF-kappa B family of transcription factors. We therefore used TPCK to examine the physiologic role of NF-kappa B during Ha-ras induced transformation, independent of another major downstream effector of Ha-ras, AP-1. Using a conditionally transformed NIH3T3 cell line, we found that TPCK (but not the control inhibitor TLME) inhibited the anchorage-independent growth of Ha-ras transformed cells, but not their anchorage-dependent growth on plastic tissue culture dishes. Likewise, TPCK reduced the ability of Ha-ras to stimulate DNA synthesis in growth factor depleted cells, but not the ability of serum to stimulate DNA synthesis in the same growth factor depleted cells. Gel shift analysis and reporter gene expression indicated that TPCK blocked Ha-ras-induced NF-kappa B activity, while only having minimal effects on Ha-ras-induced AP-1 activity. TPCK is therefore able to Inhibit the Ha-ras transformed phenotype of cells by inhibiting the transcriptional activity of NF-kappa B, while having little effect upon transcriptional activity of AP-1.

Human T lymphotropic virus type I(HTLV-I) has been implicated in various human diseases. Serum samples of 390 Brazilian Amazonians with cancer of various types were tested for HTLV-I antibodies by Gelatin particle agglutination test, Enzyme linked immunosorbent assay and Western blotting. Of 134 sera from patients with cancer of uterine cervix, 4 were positive by all the methods. Three of these were from non-transfused patients. DNA was extracted from 2 of 4 seropositive sera that gave strong reactions and were analyzed by PCR-SSCP for HTLV-I sequences. One was positive for all HTLV-I genes tested while the other one was positive for LTR and tax and negative for gag. In view of a possible pathway of the virus by sexual contact, the involvement of HTLV-I in cervical cancer warrants further studies.

Three novel isoforms of the neurofibromatosis 2 (NF2) gene transcripts generated from alternative splicing were identified from normal human brain, schwannoma and glioma tissues. The 3 novel transcripts lack exon 2, exons 2 and 3, exons 2-4, respectively. Recombinant isoform proteins encoded by those new transcripts have lost the previously reported ability to bind S-35-methionine labeled cellular proteins. Two of seven glioblastoma tissues expressed significantly high levels of the shorter transcripts whereas low grade astrocytomas expressed levels similar to those found in normal brain, suggesting that genomic mutation or aberrant alternative splicing of the NF2 gene may contribute to the progression of malignant gliomas.

UK114 is a 14 kDa protein identified in the perchloric acid soluble extract of goat liver. Anti-UK114 antibodies identify this protein as expressed by the membrane of human cancer cells. In the present study, anti-UK114 antibodies were detected in the sera of cancer patients by ELISA and by immunofluorescence tests using human cancer cell lines as a target. UK114 protein is antigenic in cancer patients. Human anti-UK114 antibodies have cytolytic effects in vitro and their presence correlates with inhibition of growth of human carcinoma cells xenografted in nu/nu mice. This finding may open new prospects for immunotherapy.

Multidrug resistance (MDR) 1 and MDR3 gene expression were examined in acute myelocytic leukemia (AML) cells from 126 Japanese patients. In 119 AML patients at diagnosis 52 were revealed to have P-gp/MDR1 (43.7%). AML cases with t(8;21) had a higher incidence of P-gp expression (13/17; 76.5%) than cases with other karyotypes (33/89; 37.1%) (p=0.0062). CD19(+) cases expressed P-gp frequently (17/25) as did CD7(+) cases (24/35). In 17 CD19(+) AML with P-gp expression, 12 had t(8;21) abnormality. In 68 AML samples examined, MDR3 mRNA was detected in 11 cases, 9 of which had the t(8;21) abnormality. The MDR3(+) t(8;21) AML samples were also positive for CD19. We analyzed P-gp expression at both diagnosis and relapse in 18 AML patients. All 11 P-gp(+) cases at relapse, in which 5 patients were P-gp negative at diagnosis, showed either t(8;21) or CD7 positivity. Our data demonstrated that expression of MDR1 and MDR3 in AML is closely associated with chromosome abnormality t(8;21) and expression of immature lymphoid antigen CD19 as well as CD7. Kasumi-1, a t(8;21) AML cell line, was demonstrated to lose P-gp by the treatment of 1,25(OH)(2)D-3, a monocyte/macropharge differentiation inducer, suggesting the possible contribution to the therapy for AML.

We determined if any extracellular matrix degradative proteases were possibly associated with metastatic potentials of human pancreatic cancer cells. Liver metastatic abilities of six human pancreatic cancer cell lines were examined in nude mice, and divided into two high (2 lines) and low (4 lines) metastatic cell lines. Of six cell lines, matrix metalloproteinases-1 (MMP-1) was overexpressed in two high metastatic cell lines: and MMP-2 was overexpressed in one high metastatic cell line. Of the four low metastatic lines, two cell lines had relatively higher mRNA levels of tissue inhibitors of metalloproteinases-3 (TIMP-3). MMP activities due to MMP-1 and MMP-2 might be positively associated with liver metastasis of pancreatic cancer cells. Moreover, expression of TIMP-3 might be partly involved in the low metastatic potentials of pancreatic cancer.

Regulation of CD44s by the c-sis oncogene product was investigated. Both CD44s protein and mRNA were comparably upregulated, consistent with some degree of transcriptional regulation; its ligand binding was also activated in confluent but not in sparse cultures of sis-transformed Balb/c 3T3 cells. CD44s was also elevated in confluent cultures of parental 3T3 cells treated with conditioned media from confluent c-sis transformants (but not from 3T3 cells); these media (but not media from ras transformants or parental 3T3 cells) contained platelet-derived growth factor (PDGF)-immunoreactive material. CD44s upregulation by these media could be partially blocked by anti-PDGF antibodies. These media also induced activation via tyrosine autophosphorylation and activation-dependent downregulation of PDGF beta-receptors. sis-transformed 3T3 cells contained low levels of PDGF receptor, but CD44 levels could still be increased in these cells by addition of PDGF. These results suggest that CD44s is upregulated in confluent cultures of c-sis-transformed cells by autocrine growth factors, including PDGF, secreted into the cell's microenvironment.

BRCA1, a major gene predisposing to breast and ovarian cancers, encodes a ring finger-containing protein. Its function is still unknown. Recently, the existence of a new structural domain called BRCT was postulated. This domain has some similarity with the 53BP1 human protein involved in p53 binding process. To test for a possible relationship between BRCA1 and p53, we compared p53 expression by immunohistochemical analysis in 29 BRCA1-associated breast cancers from 19 families, and in 200 consecutive sporadic breast cancers. We observed a prevalence of tumors with p53 positive staining in the BRCA1 population (p=0.003). In addition, p53 expression was affected by the site of the germ line mutation in the BRCA1 gene. p53 staining was found more consistently in tumors associated with mutations that lead to a truncation of BRCA1 in the ring finger region (p=0.0048). These results favor the hypothesis of a cooperation between BRCA1 and p53. Further experiments are needed to explore the full bilogical relevance of this phenomenon.

K-ras activating point mutations appear to have a role in human lung cancer, however, the prognostic significance of these abnormalities remains unclear. The aim of our work was to clarify the role of K-ras mutations as prognostic indicators in patients affected by non-small cell lung cancer (NSCLC). We studied 94 resected primary NSCLCs for K-ras mutations by the PCR-RFLP technique, followed by sequencing. K-ras activating mutations were present in 34% of tumors, a higher incidence being detected in adenocarcinomas. Comparing the impact of K-ras mutation types, we found that K-ras transversions were associated with a shorter survival in NSCLC.

Telomeres of human chromosomes consist of a repeated TTAGGG sequence, and at the terminal of this repeat sequence, the 3' strand is longer than the 5' strand. In this study, we characterized single and double stranded telomere binding proteins (ssTBPs and dsTBPs) by gel mobility shift assay and South-Western blotting assay. At least two protein components with molecular weights of 29 and 33 kDa were bound to a single stranded telomeric sequence, and also two proteins with molecular weights of about 44 kDa and 70 kDa were bound to a double stranded telomeric sequence. A competition assay demonstrated that the binding properties of ssTBPs and dsTBPs were specific to the telomeric sequence. We further cloned a ssTBP cDNA (ssTBP-1) by screening a lambda-gt11 expression library and identified ssTBP-1 as a human heterogeneous nuclear ribonucleoprotein (hnRNP) Al on the basis of cDNA sequence. We also found that the expression of the hnRNP Al gene significantly decreased during in vitro passage of human microvascular endothelial cells.

Insulin-like growth factor (IGF)-I and -II are involved in the regulation of brain development and are thought to play a pivotal role in the proliferation of gliomas. Expression of IGF-I, IGF-II, the type I and type II IGF receptor were studied in a panel of thirty glioma cell lines by Northern blotting and PCR analysis. IGF-II mRNA expression with transcripts of 4.8 and 6.0 kb was shown only in one glioma cell line (NCE-G96) and no transcripts for IGF-I, IGF-I-R and IGF-II-R could be detected by Northern analysis in total RNA. However, PCR analysis revealed signals in 19/28 cell lines for IGF-I, 27/30 for ICE-II, 19/28 for IGF-I-R and 22/28 glioma cell lines for IGF-II-R. Additional IGF-I, IGF-II, IGF-I-R and IGF-II-R PCR products were detected which might represent alternative splicing products or variants. In addition, the secretion of IGF-I and IGF-II peptides was measured by radioimmunoassay. IGF receptor status and binding characteristics were established by Scatchard analysis. Proliferation assays showed different effects of IGFs and IGF analogues on the proliferation of these cell lines. Des-(1-3)IGF-I showed an unexpected inhibitory activity on glioma cell proliferation. This may have either been due to a direct effect of the ligand for the induction of a more differentiated state refractory to its action.

In human somatic cells without the activity of telomerase, the ends of chromosomes consisting of the telomeric repeats TTAGGG progressively erode with each cell division. In germline and immortal cells telomerase activity maintains telomere length and thus compensates for the 'end-replication problem'. Progressive telomere shortening and reactivation of telomerase activity have been considered to be one of the key mechanisms in cellular senescence and immortalization. It has been shown that while most somatic cells do not have detectable telomerase activity, almost all cancers do have telomerase activity. Thus, detection of telomerase activity may have utility in the early diagnosis of cancer and may be a new target for therapeutic intervention. However, there is recent evidence that some cells of renewal tissues, such as hematopoietic cells and basal cells of the epidermis, have detectable telomerase activity. In the present study, we report detectable telomerase activity in normal human intestinal mucosa. This activity is localized to the lower third of each crypt and may be derived from intestinal stem cells. Since intestinal telomeric repeats are shorter in adults when compared to children, the telomerase activity in the intestine is insufficient to maintain telomere length but may be sufficient to provide extended proliferative capacity for such renewal tissues.

The immunodominant component of a formalinized extracellular antigen (fECA) vaccine prepared from B16 F10 melanoma cells is the melanoma-associated antigen B700. We now demonstrate that a single prophylactic intrasplenic inoculation of B700 antigen (1-10 mu g) stimulates the production of antibodies which have antiproliferative effects on B16 F10 melanoma cells in vitro. In addition, potential cytotoxic effects of splenocytes from B700 antigen inoculated mice were evaluated for two cellular immune effector functions, natural killer (NK) cell activity and lymphokine activated killer (LAK) cell activity; both activities were increased following B700 antigen inoculation. Intrasplenic injection of B700 antigen elicited an increase in the expression of the CD25 surface antigen (IL-2 R alpha) by T lymphocytes and up-regulated the expression of IL-2 R alpha mRNA. Thus both humoral and cellular cytotoxic immune responses might play roles in the decreased growth of primary tumors in B700 antigen inoculated mice and in the higher survival rate in this group of animals.

We have shown previously that various human cancer cell lines undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation or isoflavones. Here, we assessed the role of p53 gene in cell cycle and apoptosis following treatment of 11 gastric carcinoma cell lines with gamma-rays, genistein, biochanin A, or daidzein. Cell survival was measured by trypan blue staining, and apoptosis was assessed by fluorochrome staining. The rate of cell survival and apoptosis of the cells by gamma-irradiation or isoflavones did not correlate with p53 gene abnormalities. Flow cytometric measurement of DNA content demonstrated that while gamma-irradiation and genistein induced G(2) arrest, biochanin A and daidzein blocked the cell cycle of all carcinoma cells at G(1) phase. At multiple time points following irradiation, G(2) arrest was observed at 12-16 h in the wild-type and mutant p53 cell lines. Induction of p53 and p21 proteins was not observed in wild-type p53 lines after exposure to gamma-irradiation or isoflavones by Western blotting. Moreover, transfection of the wild-type p53 gene into MKN-1 cells failed to induce G(1) arrest by gamma-irradiation and genistein. Based on these results, we hypothesize that gastric cancer cells may possess a signal pathway which is different from the usual mechanisms of the p53-mediated DNA damage response in normal or hematopoietic tumor cells.

To clarify the relationship between angiogenesis and the early development of colon cancer, expression of platelet-derived endothelial cell growth factor (PD-ECGF), a known angiogenic and endothelial cell chemotactic factor, was examined in 119 human colon premalignant adenomas and colon carcinomas. Localization of PD-ECGF was assessed by immunocytochemistry in 70 nonpolypoid growth (NPG) lesions that represented 29 carcinomas (NPG carcinomas) and 41 adenomas (NPG adenomas) and 49 polypoid growth (PC) lesions that included 15 carcinomas (PG carcinomas) and 34 adenomas (PC adenomas). Simultaneously, the expression of tranforming growth factor alpha (TGF alpha) and epidermal growth factor receptor (EGFR) were examined in serial sections from these lesions. Twenty (68.9%) of 29 NPC carcinomas and 20 (48.7%) of 41 NPC adenomas exhibited positive staining for PD-ECGF, whereas 15 (100%) of 15 PG carcinomas and 27 (79.4%) of 34 PG adenomas expressed PD-ECGF. There was a statistically significant difference in the frequency of PD-ECGF expression between NPG adenomas and PG adenomas (p<0.01). In addition, the incidence of PD-ECGF expression was higher in PG carcinomas than in NPG carcinomas (p<0.05). Positive staining for TGF alpha and EGFR was detected in 14 (48.2%) and 10 (34.5%) of 29 NPG carcinomas, respectively, whereas, 17 (41.5%) and 13 (31.7%) of 41 NPG adenomas expressed TGF alpha and EGFR, respectively. A significant trend for coexpression of PD-ECGF and TGF alpha was detected in either NPG adenomas (p<0.05) or PG adenomas (p<0.01). These data demonstrate that PD-ECGF may be involved in the early stages of colon cancer development during the adenoma-carcinoma transition and additionally that angiogenesis which may be induced by PD-ECGF and/or TGF alpha could play an important role of colon cancer development.

SK-N-SH human neuroblastoma cell line was employed to test whether direct introduction of the RII beta subunit of PKA could induce growth inhibition and reverse-transformation of cancer cells. We infected SK-N-SH cells with an RII beta expression construct and the clonal line with the highest expression level was selected to examine the effect of RII beta overexpression. Elevation of RII beta was accompanied by marked reduction of RI alpha level and increase of C alpha level. Since the mRNA levels of RI alpha and C alpha were not altered by increased RII beta, the changes in the level of RI alpha and C alpha protein seem to be due to a post-transcriptional event. The RII beta-overexpressing cell line, SK-RII beta, exhibited retarded monolayer growth, decreased DNA synthesis, inability to grow in soft agar, and reduced viability in serum-free or low-serum condition as compared to parental cell, SK-N-SH. These characteristics of SK-RII beta disappeared when the RII beta expression was not fully induced. These results showed that the elevation of RII beta and/or the decrease of RI alpha is closely related to the suppression of cell proliferation and the status of transformation of SK-N-SH neuroblastoma cell line.

Histone deacetylase inhibitors such as sodium butyrate or (R)-trichostatin A {(R)-TSA; 7- [4-(dimethylamino) phenyl]-N-hydroxy-4,6-dimethyl-7-oxo-2, 4-heptadienamide} have been reported to modulate the proliferation and differentiation of certain cell types. In this study, we analyzed the effects of these agents on KM12 human colon carcinoma (HCC) cells in culture. We found that (R)-TSA induced cell flattening, inhibited anchorage-dependent and anchorage-independent growth, increased the level of the differentiation marker carcinoembryonic antigen, and increased the expression of gelsolin, a candidate tumor suppressor, in these HCC cells. Cells treated with (R)-TSA for 3 h exhibited a high degree of histone (primarily H4) acetylation. (R)-TSA exerted these effects at concentrations in the range between 0.1 to 1 mu M that are at least 1,000 times lower than those required to achieve similar effects by n-butyrate. Trichostatin C, which exhibits some histone deacetylase inhibitory activity but not the analogs (S)-TSA or (R)- or (S)-trichostatic acid, which lack histone deacetylase inhibitory activity, also showed some growth inhibitory activity. These results indicate that histone deacetylase inhibitors may lead to suppression of the transformed phenotype and enhanced differentiation in the KM12 HCC cells.

The monoclonal antibody (P-3 mAb) and the rabbit immune sera (RIS) recognizing a 14 kDa perchloric acid soluble protein extracted from goat liver (UK 114), locate this antigen on the cell membrane of several human cancer cell lines. UK 114-positive cells (e.g. HT 29 and KATO VI cells) undergo antibody-dependent cytolysis bl vitro. In nu/nu mice bearing xenografted HT 29 cells, tumor growth was markedly impaired by peri-tumoral injection of anti-UK 114 antibodies. These experiments suggest that human tumors expressing UK 114 over the cell membrane may undergo antibody-mediated cytolysis and growth control.

A new continuous cell line from a human malignant carcinoid of the small intestine (KRJ-I) was established. The cells showed morphological and immunocytochemical features of the tumor of origin and expressed estrogen receptors. The cells are growing as a suspension, forming multicellular aggregates and spheroids. Electron microscopy confirmed the presence of neuroendocrine granules. Dose-dependent growth inhibition was observed after incubation with 5-azacytidine. Cytogenetic analyses of the tumor of origin, the cell line KRJ-I and a liver metastasis KRJ-II revealed clonal tetraploidy and clonal loss of the Y-chromosome and chromosome 19.

Retinoids restore normal cell growth and differentiation in malignant cells and are considered as potential therapeutic agents. Before using retinoids for the treatment of pancreatic cancer it is important to determine the expression of nuclear retinoid receptors, which mediate most actions of retinoids on gene expression in normal and malignant tissues. Digoxigenin-labeled antisense riboprobes of retinoic acid receptors (RARs) alpha, beta, and gamma, and retinoid X receptor (RXR) alpha were used for in situ hybridization to histological sections of-specimens from 24 human pancreatic carcinomas, 20 of which also contained adjacent normal tissue. All four receptors were detected in adjacent normal pancreatic tissue specimens and RAR-alpha, RAR-gamma, and RXR-alpha were also detected in all pancreatic carcinoma specimens. In contrast, RAR-beta mRNA transcripts were detected in only 67% of the malignant tissues and when expressed, the level of expression was significantly lower than that of the corresponding adjacent normal tissues. Decreased RAR-beta gene expression was especially noted in moderately- and poorly-differentiated cancers. These findings suggest that selective decrease or lass of RAR-beta gene expression in certain pancreatic carcinomas in vivo might be associated with the development or progression of pancreatic cancer.

We utilized fluorescent-labeled primers and modified the telomeric repeal amplification protocol (TRAP) to assess telomerase activity during the process of in vitro establishment in four different leukemia cell lines. We demonstrate that three of four leukemia cell lines had elevated telomerase activity, shortened telomeres, and the appearance of either dicentric chromosomes or acentric fragments. By contrast, one leukemia cell line (HAL-01) had high levels of telomerase activity, stable telomeres, but no obvious additional cytogenetic rearrangements. These experiments indicate that there may be several pathways involving telomerase activity in the establishment of leukemia cell lines.

The epidermal growth factor (EGF) receptor is activated by EGF and other EGF-like growth factors, including heparin-binding epidermal growth factor-like growth factor (HB-EGF). We characterized the biological actions of HB-EGF in PANC-1 and COLO-357 human pancreatic cancer cell lines, and determined whether the presence of HB-EGF in human pancreatic carcinomas correlates with patient survival. HB-EGF enhanced the growth of both cell lines in a dose-dependent manner, with a potency that was generally similar to that of EGF and transforming growth factor-alpha (TGF-alpha). HB-EGF also readily induced tyrosine phosphorylation of the EGF receptor in these cells. Immunohistochemical analysis of 47 pancreatic cancer tissues revealed the presence of HB-EGF immunoreactivity in the cancer cells in 50% of the tumors. However, the presence of HB-EGF was not associated with a statistically significant decrease in the post-operative survival period. Furthermore, coexpression of HB-EGF and the EGF receptor was not associated with shorter patient survival. These findings suggest that HB-EGF activates the EGF receptor in human pancreatic cancer cells, but that it is not involved in enhancing the biological aggressiveness of this malignancy in vivo.

We report the development of an immuno-lectin-enzymatic assay (CA83.4) with the purpose of quantifying serum glycoproteins bearing Tn determinant (GalNAc alpha-O-Ser/Thr). An anti-Tn monoclonal antibody (83D4) is bound to the solid phase in order to capture glycoproteins. After the addition of a test sample, we used biotinylated isolectin B4 from Vicia villosa and avidin-peroxydase to act as a detection system. The linear relationship between CA83.4 determinations and the serum dilutions, the reproducibility of the dosage in intra- and inter-assay, and the specificity of the test for the N-acetylgalactosamine residue in a-glycosidic O-linkages, demonstrated the reliability of this trial. Self-recognition of Vicia villosa B4 molecules (K-D: 0.73x10(-6) M determined using biosensor technology) could determine an additional step of signal amplification in this assay. Using 0.25 units/ml of CA83.4 antigen as the cut-off level, higher values were found in 25/49 patients with breast cancer, 8/13 with colorectal carcinoma, 3/11 with lung cancer, but in none of the 49 patients with non-malignant diseases nor in 97 healthy controls. This first report on soluble Tn-glycoprotein detection assays suggests that Tn-glycoproteins are specific serological tumor markers and we believe that they could represent a valuable tool in the diagnosis of cancer.

Human fibroblast cells must overcome both the M1 and the M2 stages of cellular senescence to immortalize, at which point cells almost always express telomerase activity. The human papillomavirus (HPV) oncoproteins, HPV-16 E6 and E7, can block the progression to senescence in fibroblasts by associations with p53 and pRb, respectively. Human mammary epithelial (HME) cells require only HPV-16 E6 to bypass M1, suggesting that pRb may not have a direct role in HME cells senescence. In the present report, we show that only wild-type HPV-16 E6 allows complete degradation of p53, immortalization and reactivation of telomerase activity in HME cells. These results suggest that the ability of HPV-16 wild-type and mutant E6 proteins to degrade p53 in intact HME cells and keratinocytes does not completely correlate with their ability to degrade p53 in a cell-free system. This discrepancy between in vitro and in vivo p53 degradation may be biologically significant and may provide insight into the susceptibility of certain human cells and tissues for reactivation of telomerase and immortalization.

The extent- and cell cycle specificity of apoptotic cell death were studied in human leukemia HL-60 cells treated with 1.25-20 mu M etoposide, 0.125-2.0 mu g/ml dactinomycin, 12.5-200 mu M 3-deazaadenosine (c(3)Ado) and 10-50 Gy gamma-irradiation. Flow cytometry was used to measure the fraction of apoptotic cells and determining their cell cycle position. With all agents the extent of apoptotic cell death showed a clear dose- and time-dependency. Specific apoptosis of S-phase cells was found in cultures treated with 1.25-2.5 mu M etoposide. At 5-20 mu M etoposide, 0.125-2.0 mu g/ml dactinomycin and 50-200 mu M c(3)Ado cells from all cell cycle phases responded by apoptosis. Exposure of the cells to 10 Gy gamma-irradiation resulted in apoptosis of G(2)+M-phase cells. At 50 Gy cells from the S- and G(0)/G(1)-phases also entered apoptosis. Our findings indicate that the cell cycle specificity of an anti-cancer agent initiated apoptotic response depends upon the drug/irradiation dose and exposure time used. This demonstrates that the resistance of subpopulations of cells to apoptosis is relative.

We analyzed chromosomal alterations in primary lung tumours and peripheral blood lymphocytes (PBLs) from 10 lung cancer patients (nine with non-small cell lung carcinoma and one with small cell lung carcinoma) to determine whether there were shared chromosomal changes in the normal and diseased tissue of these cases. This study revealed that each paired sample had at least three chromosomes and two chromosomal regions in common for structural rearrangements. The chromosomes most frequently found structurally altered in paired analysis were 1 and 3 (60% each), 5 (50%), 6, 7, and 9 (40% each), and 14 (30%). Chromosome region 3p13-3p21 was structurally rearranged in both the normal and tumour tissues of three patients. Chromosome 3 was structurally rearranged in all ten tumours. The chromosome arms most commonly affected in the tumours were 3p (nine times), 9p and 5q (eight times each), Ip and 7q (six times each), 10q and 11q (five times each), 14q and 6q (four times each). The most frequently affected chromosomal regions in these tumours were (in decreasing order) 9p23-p24, 3p21-3p13, 5q11, 1p34, 7q22, and 11q13. Frequent polysomy of 7 and 12 and loss of D-group chromosomes were also observed in the tumours analyzed. Comparing the changes found only in tumours with those found in both PBLs and tumours was helpful in shedding some light on the probable sequence of genetic events leading to lung cancer. This investigation also offered compelling evidence that genomic instability at the chromosomal level in PBLs corresponds with the genetic changes observed in tumours indicating that PBL analysis can help identify the early chromosomal changes in lung cancer. PBL chromosomal analysis thus has a promising future in the genetic analysis of lung cancers.

In order to establish prognostic indicators of poor clinical evolution in colorectal cancer, we have studied p53 abnormalities and K-ras mutations in 65 patients affected by colorectal carcinoma who had undergone radical surgery. A single event of p53 protein overexpression or p53 mutation, but not K-ras mutation, was significantly prevalent in recurrent tumors. A double event of K-ras mutation and p53 protein overexpression was prevalent in patients showing Dukes' stage C, but showed a lack of prevalence in patients who recurred. The presence of p53 protein overexpression does not assure an underlying mutation in colorectal carcinoma.

The development of the breast evolves through progressive formation of lobular structures. Lobules type 1 (Lob 1) are the most undifferentiated ones; they evolve to lobules type 2 (Lob 2), and those to type 3 (Lob 3), in response to mainly ovarian hormones. Although estradiol and progesterone act by binding to their specific receptors, their content in these structures and how they correlate with proliferative activity are not known. Normal breast tissues obtained from 40 reduction mammoplasties performed for cosmetic reasons in women free of mammary pathology were processed for light microscopy. Paraffin sections were immunoreacted with antibodies against estrogen receptor (ER), progesterone receptor (PgR), and proliferating cell nuclear antigen (PCNA). The number of cells positively labeled with each one of the three antibodies was quantitated in Lob 1, 2 and 3, and expressed as a percentage of the total number of cells. ER were positive in Lob 1 (21%) and Lob 2 (10%), but negative in Lob 3. PgR were positive in Lob 1 (66%) and 2 (68%), decreasing in Lob 3 (31%). The highest percentage of PCNA positive cells were found in Lob 1 (40%), decreasing in Lob 2 (33%) and Lob 3 (11%). These results indicate that the normal breast epithelium contains ER and PgR, but the former is at higher levels in Lob 1, which also expresses greater proliferative activity as demonstrated with PCNA.

Although several oncogenes and tumor suppressor genes have been suggested to be of relevance for the development of oral cancer, it is likely that additional genes are involved in this complex process. Therefore, in an attempt to isolate such genes, the aim of this study was to investigate changes in gene expression in human buccal carcinoma cells as compared to normal buccal epithelial cells, and identify mRNA overexpressed in the carcinoma cell line. The method of differential display of mRNA was used to isolate differentially expressed genes (Liang P et al, Science 257:967-971, 1992). A key step of this method, a polymerase chain reaction amplification, was optimized in terms of choice of thermostable DNA polymerase, annealing temperature, molar ratios and concentrations of primers. The comparative analysis of expression in tumor and normal buccal epithelial cells led to the isolation of three different mRNAs overexpressed in human oral carcinoma cells, as confirmed by Northern blot analysis. Cloning and sequence analysis revealed that these genes, which were termed OTEX as in Oral Tumor EXpressed, included a novel, previously not characterized, human gene, OTEX-1. OTEX-2 was identical to the gene coding for the L26 ribosomal protein, a protein known to be overexpressed also in other tumor cell types. OTEX-3 showed a perfect match to a sequence isolated during the human genome sequencing project, with a hitherto unknown function.

We conducted transient reporter gene expression assays to show that the wild-type but not the mutant-type p53 protein positively regulates the expression of the human H-ras gene. This activation of expression is mediated through a specific p53-binding DNA element located in the first intron of the H-ras gene. This element is similar to a previously identified p53-binding element. Without this element, wild-type p53 represses the H-ras promoter, as do several p53 mutants. The repression function is lost when the N-terminal 81 amino acids of the p53 protein are deleted, suggesting that the N-terminus is required for repression. Therefore, p53 seems to regulate the H-ras through both positive and negative mechanisms.

Human pancreatic ductal adenocarcinomas overexpress the epidermal growth factor (EGF) receptor. Betacellulin is a mitogenic polypeptide that binds and activates this receptor. To determine whether betacellulin has a role in human pancreatic cancer, we studied its expression in cultured human pancreatic cancer cell lines and in normal and cancerous pancreatic tissues. Five of 6 pancreatic cancer cell lines expressed the 3 kb betacellulin mRNA moiety, T3M4, MiaPaCa-2 and COLO-357 cells exhibiting the highest expression levels. EGF, heparin-binding EGF-like growth factor (HB-EGF), and basic fibroblast growth factor (bFGF) increased betacelullin mRNA levels. Only 2 of 15 normal samples and 1 of 10 cancer samples failed to exhibit the betacellulin transcript. Densitometric analysis of the autoradiographs revealed a 7.5-fold increase in betacellulin mRNA levels in the cancer tissues by comparison with the normal tissues. By in situ hybridization, the duct-like cancer cells exhibited many betacellulin mRNA in situ hybridization grains. These findings indicate that human pancreatic cancer cells express betacellulin in culture and in vivo, and suggest that this EGF-like ligand may participate in aberrant autocrine and paracrine activation of the EGF receptor in human pancreatic cancer.

Intracerebral injection of human polyomavirus, JCV, into neonatal hamsters causes tumors of,glial origin. HJC is an established cell Line derived from a JCV-induced mixed hamster brain tumor with astrocytic and ependymal components. Flow cytometric and immunohistochemical analysis of HJC suggests that it is comprised of a mixed population of cells all of which contain the JCV early protein, T-antigen, in the nuclei. Five individual clonal lines, called HJC-15a to HJC-15e, were isolated by limiting dilution and were found to exhibit distinct morphological characteristics with 25-30% variation in their sizes. It was evident that each clone has unique growth rates, doubling times, and cell cycle parameters with different G(1), S, and G(2) phase times. All clonal cells showed the presence of the JCV early protein in the nucleus. Of interest was the observation from immunoprecipitation and Western analysis indicating qualitative and quantitative differences in the T-antigen isoforms produced in these cells. Similar to the parental clone, HJC-15b produced two distinct forms of JCV T-antigen isoforms, 88 kDa and 92 kDa proteins. In addition, HJC-15c was able to produce a 23-25 kDa protein which was recognized by anti-T-antigen antibody. The activity of cyclin-dependent kinases, in particular cdc2, was higher in HJC-15c than in the other cell lines. The data presented herein indicates that glioblastomas induced by viral T-antigen expression are composed of a multitude of distinct cells that possess a variety of different characteristics.

Retinoids can inhibit the growth and modulate the differentiation of a variety of tumor cell types in vitro and in vivo. All-trans retinoic acid (ATRA) and N-(4-hydroxyphenyl)retinamide (4HPR) are currently being evaluated in clinical trials for their potential use in cancer chemoprevention and therapy. We compared the effects of these retinoids on 10 human cervical carcinoma cell lines. Four of the 10 cell lines showed dramatic morphological changes and the other 5 exhibited decreased cell density after treatment with 10 mu M 4HPR, whereas few changes were induced by 10 mu M ATRA. Cell rounding and detachment were also observed in four of the cell lines. An analysis of DNA from both detached and attached cells after retinoid treatment has demonstrated the formation of a DNA ladder after electrophoresis in agarose gels, which indicated that some of the cell lines had undergone apoptosis. Induction of DNA fragmentation by 4HPR but not by other retinoids (ATRA, 13-cis-RA, and 9-cis-RA) was further evidenced as early as 24 h after treatment by a quantitative assay based on the degradation of [H-3]-thymidine-labeled DNA. Ln addition, morphological changes of nuclei associated with apoptosis such as chromatin condensation were observed by propidium iodide staining of the nuclei after 4HPR treatment. These results demonstrate that 4HPR causes apoptosis in several cervical carcinoma cell lines and that it is more potent in this effect than ATRA or other RA isomers.

Chronic administration of 17 beta-estradiol to male Syrian hamsters for at least six months induces estrogen-dependent kidney tumors, which express high levels of c-fos mRNA compared to surrounding renal or control tissues. We have investigated the cellular localization of c-Fos oncoprotein in these tissues and studied the estrogenic regulation of c-fos mRNA in kidney and in H-301 cells, a cell line derived from primary hamster kidney tumors. Immunocytochemical analyses showed that levels of c-Fos protein were high in estrogen-dependent tumors. To study the early events in this model, hamsters were treated with 17 beta-estradiol for only 15 days. At this time, well before the appearance of tumors, c-Fos protein was concentrated in interstitial capillaries, arteries, and podocytes of the glomerulus, but not in renal tubular epithelium, and this pattern was not appreciably changed from controls. To study the regulation of c-fos that led to the altered expression in tumor vs. kidney, total RNA was isolated from kidneys of Syrian hamsters 3-48 h after treatment with single injections of either 17 beta- or 17 alpha-estradiol, 17 alpha-ethinylestradiol, the steroidal antiestrogen ICI 182,780, or 17 beta-estradiol plus ICI 182,780. Similar studies were carried out on H-301 cells grown in D-MEM/F-12 medium and charcoal-stripped serum. The increases in c-fos mRNA levels in H-301 cells but not kidney were elicited by classical estrogen receptor-mediated processes. In H-301 cells, c-fos levels were increased four-fold over controls after 3 h of 17 beta-estradiol treatment and this induction was suppressed by antiestrogen treatment. In hamster kidneys, c-fos levels were increased about two-fold by 17 beta-estradiol, but this induction was not affected by antiestrogen treatment. In H-301 cells but not in hamster kidneys, 17 alpha-ethinylestradiol induced c-fos. 17 alpha-estradiol was more potent than 17 beta-estradiol in the induction of c-fos in hamster kidney but was a poor inducer in H-301 cells. These studies are consistent with regulation of c-fos expression in H-301 tumor cells by an estrogen receptor-mediated mechanism. But in hamster kidney, c-fos expression does not appear to be regulated by receptor-mediated pathways. We propose that it may be induced by estrogen metabolites.

Thirty samples from 19 patients with synovial sarcoma were analyzed cytogenetically after short-term culturing. Thirteen samples were from primary tumors, 11 from local recurrences, and six from distant metastases. All samples showed the characteristic aberration t(X;18)(p11;q11) or variants thereof; 23 samples had additional numerical and/or structural changes. Including the present cases, chromosome aberrations have been reported in 74 synovial sarcomas, 50 of which have had secondary aberrations in addition to t(X;18). No secondary structural aberration was recurrent. The most common numerical changes were +7, +8, +12 (10 cases each), -3, +9, +21 (7 cases each), +2, -14, -17 (6 cases each), +4, -11, +15, and -22 (5 cases each). Unbalanced stuctural aberrations led to loss of 3p and 17p in six cases, each with loss of bands 3p21 and 17p13, respectively, in common. Most monosomies and trisomies seemed to occur at similar frequencies in primary, recurrent, and metastatic tumors. The only exceptions were +2, which was never seen in a primary tumor, and +8, which was never found in any metastatic lesion.

Conventional cancer therapies such as surgery, chemotherapy and/or radiotherapy, are not always successful in providing long-term survival for cancer patients. One of the major problems with conventional cancer therapies is their inability to eradicate residual/metastatic tumor cells that are resistant to therapy. Therefore, it is necessary to develop new methods for treating such cancer cells in order to improve the clinical outcome of these patients. Despite antitumor effector mechanisms working against cancer cells in the host's body, tumor-cell-induced immunosuppression and or antigenic modulation by the tumor cells often help tumor cells escape host defense mechanisms. Therefore, one approach for treating residual cancer would be to enhance the host's own immunological/antitumor defense mechanisms. Immune cells that have a significant role in mediating antitumor responses include: T lymphocytes; natural killer (NK) cells; macrophages; and B lymphocytes. The ability of these immune cells to effectively destroy malignant cells is carefully governed by chemical mediators in the form of proteins otherwise known as cytokines. Many cytokines (interleukins, interferons, and tumor necrosis factor) have been shown to enhance in vitro and in vivo effector cell antitumor cytotoxic activities. Utilization of cytokines in conjunction with effector cells can also mediate significant antitumor responses in both animal models and cancer patients. One of the major problems associated with systemic treatment with cytokines is the development of dose limiting toxicities. Currently, attempts to reduce this problem include developing techniques to allow for the preferential release of cytokines in proximity to the tumor cell. In this regard, effector cells or tumor cells that have been genetically engineered to secrete cytokine(s) may be useful in localizing an immune response, preferably at the tumor site. Clinical trial using cytokine gene transfected cells for treating cancer are currently under investigation. With the availability of recombinant lymphokines and with our ability to genetically modify effector cells and tumor cells this hopefully will allow us to improve current therapeutic modalities for treating cancer.

The urokinase-type plasminogen activator contributes to the invasive phenotype of tumor cells by facilitating extracellular matrix hydrolysis. However, the molecular mechanism(s) responsible for urokinase overexpression remains unclear. The current study was undertaken to determine the role of the p53 tumor suppressor gene in the regulation of urokinase expression. Transient cob transfection of a urokinase-producing cell line with a wildtype p53 expression vector and a CAT reporter driven by the urokinase promoter led to a dramatic reduction in CAT activity. Conversely, urokinase promoter activity was increased in cells transiently transfected with a p53 expression vector mutated at codon 175. To determine if the endogenous urokinase gene was modulated by p53, cells were stably transfected with a mutated p53-bearing expression vector. An increased level of urokinase mRNA was apparent in pooled mutant p53-overexpressing clones. These studies argue for a role of the p53 tumor suppressor gene in the regulation of urokinase expression.

Cellular mortal and immortal phenotypes as defined by the limited and the infinite capacity of cells to divide are the characteristics of normal and cancerous cells in culture. Numerous strategies that have been employed to understand the mechanism(s) of normal as well as tumor cell growth have revealed that these are genetically controlled, however, the genes and the synchronized regulations remain largely undefined so far. The present report reviews the identification of mortalin, a novel member of murine hsp70 family of proteins, as a gene involved in pathways that determine divisional phenotype of cells in vitro. In the present study, the anti-proliferative activity of mortalin is demonstrated also in human skin fibroblasts (TIG-73PD) by microinjection of anti-mortalin antibody. Furthermore, studies on the mortalin immunofluorescence patterns in SV40-immortalized pre-crisis and post-crisis human cells have revealed that the change in the intracellular distribution of mortalin is linked to the change in the divisional phenotype of cells. Thus, the studies to resolve the molecular basis of association of the cytosolically distributed form of mortalin with cellular mortal phenotype would be important in understanding of the mechanism(s) that determine replicative potential of cells in culture.

The extracellular cGMP levels or the ratio between extracellular levels of cGMP and cAMP (cGMP(ex)/cAMP(ex)) have been proposed as tumor marker for premalignant and malignant diseases of the uterine cervix. More than 50% of cervical cancers occur in premenopausal women and detailed information about hormonal and drug effects on the extracellular levels of cyclic nucleotides is of importance. In the present study we have investigated the effect of progesterone (0.1-100 mu M), theophylline (1-1000 mu M), probenecid (0.1-100 mu M) and verapamil (0.1-100 mu M) on cGMP(ex)/cAMP(ex) of C4-I cells (a human cell line derived from a squamous carcinoma of the uterine cervix). Within 30 min progesterone caused a concentration-dependent elevation of cGMP(ex)/cAMP(ex), whereas the other compounds had no marked effect. Identical results were obtained for C4-I cells in monolayer and in suspension. The effects were explained by the observation that progesterone stimulated cGMP efflux, but inhibited the cAMP efflux. The other compounds inhibited the export of both nucleotides to a similar degree. The present data suggest that progesterone affects the export of cyclic nucleotides in non-genomic manner and may hamper the interpretation of cGMP(ex)/cAMP(ex) in the luteal phase in premenopausal women with cancer of the uterine cervix.

Marek's disease virus (MDV) is one of the most oncogenic herpesviruses and the only cancer virus that has a successful vaccine. Based on the short-latency and polyclonal nature of the tumors, it is likely that MDV encodes its own oncogenes. Recent studies have provided insights into possible MDV genes involved in oncogenesis. We describe the characterization of two gene families encoded by the BamHI-H and I-2 fragments. The BamHI-H and I-2 fragments, located contiguously in the IR(L) region, are unique to oncogenic (serotype) MDV and consistently expressed in MDV-transformed tumors and T-cell lines. The structural alteration of the BamHI-H fragment, specifically the expansion of a 132 bp repeat, has been correlated with the attenuation of the oncogenicity of MDV. Several open-reading frames, some resulting from alternative splicing, can be identified. One of the open-reading frames, BHa, encodes a 7 Kd polypeptide. BHa shares limited homology with a T-cell lymphoma oncogene TLM and stimulates extended growth of chicken embryo fibroblasts, when transfected into the cell. The other gene implicated in latency or oncogenesis is Meg whose coding sequences span portion of the BamHI-I-2 and the Q(2) fragment. Meq has an interesting structure, resembling a chimera between Jun/Fos oncoproteins and WT-1 tumor suppressor protein. It contains a bZIP (basic leucine zipper) domain and a long proline-rich domain. The proline domain has trans-activating function, whereas the bZIP domain allows Meq to dimerize with a variety of transcriptional factors and expands the enhancer repertoire it acts on. As neoplastic transformation usually requires the complementation of multiple oncogenes, BHa and Meq could be two of the proteins participating in this process.

The longer survival of ovarian cancer patients after immunostimulation has been connected with the induction of an anti-tumor activity triggered by cellular and humoral immune responses. Our interest was to study the long-term influence on the immune system in relation to the various levels of the HAMA response and the disease stage. The immunological profile was evaluated by regularly performing phenotyping and functional analysis of peripheral blood lymphocytes (PBL). We report the statistical analysis of immunological data obtained in a study with 77 ovarian cancer patients examined over a period of up to 28 months. The results demonstrate that these immunological data are important for monitoring cancer patients under immunotherapy whereas they provide no prognostic significance.

Expression of human epidermal growth factor (EGF) and matrix metalloproteinase-9 (MMP-9/gelatinase B) was examined immunohistochemically in 62 cases of surgically resected esophageal carcinomas, and the correlation between EGF expression and the proliferative activity of the tumors was studied by analysing the number of proliferating cell nuclear antigen (PCNA)-positive cells. Expression of EGF and MMP-9 was observed in 16 (38.1%) and 18 (42.9%) of the 42 superficial carcinomas and 8 (40%) and 14 (70%) of the 20 advanced carcinomas, respectively. The differences in the MMP-9 expression between the superficial carcinomas and the advanced carcinomas was significant (p<0.05). The synchronous expression of EGF and MMP-9 was observed in 15 (24.2%) of 62 carcinomas, i.e. 62.5% of the 24 EGF-positive tumors expressed MMP-9, but there was no statistically significant correlation between the expression of EGF and MMP-9. The relationships between EGF expression and tumor proliferative activity and prognostic factors were investigated. The PCNA grades were significantly higher in tumors with EGF-positive than those with EGF-negative expression (p<0.05) and the EGF expression showed a good correlation between the expression of MMP-9 and vascular invasion (p<0.01). The expression of MMP-9 was stronger in the advanced than the superficial carcinomas and there was a good correlation with vascular invasion (p<0.01). In a follow-up study of 55 patients, those with tumor that expressed MMP-9 or had a high PCNA grade showed a poor prognosis. Taken together, these observations suggest that both EGF and MMP-9 participate in the invasive phenotype in human esophageal carcinoma, but the expression of EGF is not directly related to the expression of MMP-9. Additional growth factors and cytokines may be involved in regulation of MMP-9 expression in this carcinoma.

The epidermal growth factor (EGF) receptor (EGFR) is activated by EGF and other EGF-like growth factors, including amphiregulin (AR). We characterized the localization and mitogenic action of AR in T3M4 and COLO-357 human pancreatic cancer cell lines and determined whether the presence of AR in human pancreatic cancers correlates with patient survival. Both T3M4 and COLO-357 cells were found to be extremely sensitive to AR, one-half maximal stimulation occurring at a concentration of 70 and 50 pM, respectively. The magnitude of the stimulatory effect was greater with AR than with EGF. Both cell lines exhibited AR immunostaining, which was present in a variable manner in the cytoplasm, nucleus and nucleoli. Immunohistochemical analysis of 62 pancreatic cancer tissues revealed the presence of nuclear and/or cytoplasmic AR immunoreactivity in the cancer cells. Cytoplasmic, but not nuclear localization of AR in the pancreatic cancer cells was associated with a statistically significant decrease in the post-operative survival period. The presence of EGFR alone in the cancer cells did not correlate with decreased survival, whereas coexpression of cytoplasmic AR and EGFR was associated with shorter survival. Distant metastases did not always exhibit cytoplasmic AR immunoreactivity. These findings point to the existence of an EGFR: AR autocrine loop in human pancreatic cancer which may contribute to its biological aggressiveness.

The response of a rodent fibrosarcoma to photodynamic therapy (PDT), radiotherapy (RT) or combined PDT and RT was examined. PDT used intravenous polyhaematoporphyrin (PHP) and interstitial illumination with 630 nm light (300 or 400J). RT was given as an external beam of gamma-rays (15, 20 or 25 Gy). Both PDT and RT induced a significant delay in tumour growth when applied alone. PHP given alone, without subsequent illumination, did not alter the response of the tumour to RT. The effect of combining PDT and RT was dependent on the dose of light administered, but not, in most cases, on either the radiation dose or the sequence in which the two treatments were given. Using 300J of light, all combined treatments produced additive tumour growth delays. In contrast, sub-additive tumour responses were observed following most combined treatments using 400J of light.