Expression of the SART-1 Antigens in Uterine Cancers

We recently reported that the SART-1 gene, encoding the SART-1₂₅₉ tumor antigen which is recognized by HLA-A26-restricted cytotoxic T lymphocytes (CTLs), is expressed in the cytosol of squamous cell carcinomas and adenocarcinomas. The present study deals with the expression of SART-1₂₅₉ and SART-1₈₀₀ antigens in uterine cancers. The SART-1₂₅₉ antigen was detected in the cytosol fraction of 4 of 8 uterine cancer cell lines, 24 of 74 (32%) uterine cancer tissues, 0 of 7 uterine myomas, and 0 of 5 non-tumorous uterine tissues. The SART-1₈₀₀ antigen was expressed in the nuclear fraction of all the uterine cancer cell lines, 41 of 74 (55%) uterine cancer tissues, 0 of 7 myomas, and 3 of 5 non-tumorous uterine tissues. The SART-1₂₅₉⁺ uterine cancer cells were recognized by HLA-A24 restricted and SART-1 specific CTLs. Therefore, SART-1₂₅₉ antigen could be an appropriate vaccine candidate for a relatively large number of uterine cancer patients.     

Frameshift Mutations and a Length Polymorphism in the hMSH3 Gene and the Spectrum of Microsatellite Instability in Sporadic Colon Cancer

Mutations in the hMSH3 gene in sporadic colon cancer with microsatellite instability (MSI) were investigated, since several mismatch repair genes were known to be mutated in cancers with MSI, but only deletions in the (A)₈ region in the hMSH3 gene have been reported. We also analyzed the relationships between hMSH3 mutations and the spectrum of MSI. We screened MSI in 79 sporadic colon cancer samples using mono- and dinucleotide repeat markers and the samples with MSI were further analyzed for tri- and tetranucleotide repeat instability and mutations in the hMSH3 gene by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Five (6%) out of 79 tumors were MSI-H and 15 (19%) were MSI-L. Two MSI-H tumors showed insertion in the (C)₈ region in the hMSH6 gene and one tumor showed insertion and deletion in the (A)₈ region in the hMSH3 gene, and two of the three above tumors showed MSI in tri-and tetranucleotide repeats. One MSI-L tumor showed somatic alteration in a 9-bp repeat sequence in hMSH3. No frameshift mutations were found in the (A)₇ and (A)₆ regions in hMSH3. Thus, we confirmed that the (A)₈ region in hMSH3 is a hot spot and mutations in the (A)₇ and (A)₆ regions in hMSH3 are not common. The hMSH3 mutation may enhance genomic instability in some colorectal cancers.     

Expression of the SART-1 antigens in uterine cancers.

We recently reported that the SART-1 gene, encoding the SART-1(259) tumor antigen which is recognized by HLA-A26-restricted cytotoxic T lymphocytes (CTLs), is expressed in the cytosol of squamous cell carcinomas and adenocarcinomas. The present study deals with the expression of SART-1(259) and SART-1(800) antigens in uterine cancers. The SART-1(259) antigen was detected in the cytosol fraction of 4 of 8 uterine cancer cell lines, 24 of 74 (32%) uterine cancer tissues, 0 of 7 uterine myomas, and 0 of 5 non-tumorous uterine tissues. The SART-1(800) antigen was expressed in the nuclear fraction of all the uterine cancer cell lines, 41 of 74 (55%) uterine cancer tissues, 0 of 7 myomas, and 3 of 5 non-tumorous uterine tissues. The SART-1(259)+ uterine cancer cells were recognized by HLA-A24 restricted and SART-1 specific CTLs. Therefore, SART-1(259) antigen could be an appropriate vaccine candidate for a relatively large number of uterine cancer patients.     

Serine Proteinase Inhibitor 9 Can Be Recognized by Cytotoxic T Lymphocytes of Epithelial Cancer Patients

Serine proteinase inhibitor 9 (PI–9) inhibits granzyme B-mediated apoptosis and interleukin–lβ-converting enzyme activity. In this study, we report that the PI–9 gene encodes antigenic epitopes recognized by the HLA-A24–restricted and tumor-reactive cytotoxic T lymphocytes (CTLs) of epithelial cancer patients. Screening of an autologous cDNA library using a CTL line recognizing HLA-A24⁺ tumor cells resulted in the isolation of a cDNA, which had an identical coding region to the previously described PI–9 genes. PI–9 gene was expressed in approximately three-fourths of epithelial cancer cell lines and all leukemic cell lines tested. It was also expressed in normal peripheral blood mononuclear cells (PBMCs), but not in a normal fibroblast cell line. CTL sublines contained T cells capable of recognizing the PI–9_292–300 and PI–9_348–356 peptides among 13 different peptides having the HLA-A24 binding motifs. These two peptides were recognized by the CTL line in a dose-dependent and HLA class-I-restricted manner, and also possessed the ability to induce HLA class I-restricted and tumor-reactive CTLs in PBMCs from HLA-A24⁺ cancer patients. These results demonstrate that PI–9 is recognized by HLA class I-restricted and tumor-reactive CTLs of epithelial cancer patients.     

Inhibition of Cell Attachment, Invasion and Metastasis of Human Carcinoma Cells by Anti-integrin β1 Subunit Antibody

We investigated the expression of β₁ integrins in human carcinoma cell lines, and the anti-metastatic and anti-invasive effects of a newly established anti-human β₁ subunit monoclonal antibody designated NCC-INT-7. All the examined carcinoma cell lines expressed β₁ integrins upon immunoblot analysis. NCC-INT-7 completely inhibited the adhesion of carcinoma cells to laminin, fibronectin, collagens and acetone-fixed tissues including lung, liver and brain. In an in vitro invasion model, NCC-INT-7 inhibited the invasion of human bladder carcinoma cell line T24 and human gastric carcinoma cell lines TMK-1, MKN-45 and MKN-74 through an artificially reconstructed basement membrane. In an in vivo nude mouse peritoneal dissemination model using MKN-45 and TMK-1, NCC-INT-7 significantly reduced the number of tumor nodules in the mesentery. In an in vivo nude mouse liver metastasis model using a serially transplantable human colonic carcinoma, COL-2–JCK, NCC-INT-7 significantly reduced the number of tumor nodules in liver. These results indicate that β₁ integrins play an important role in the tissue attachment, migration, invasion and metastasis of human carcinoma cells, and that this new monoclonal antibody is useful for studies aimed at prevention of metastasis.     

Mutational Analyses of Multiple Target Genes in Histologically Heterogeneous Gastric Cancer with Microsatellite Instability

It has been recognized that gastric cancer often shows histological heterogeneity in a single tumor. Although microsatellite instability (MSI) has been reported in gastric cancer, the significance of genomic instability in gastric cancers with histological heterogeneity within a single tumor has never been addressed. We investigated MSI at 8 microsatellite loci in 40 normal/tumor DNA pairs from 20 gastric cancers with histological heterogeneity. Six of 20 patients (10 DNAs of 40 tumor DNAs) had severe MSI in more than 3 loci. Four of the MSI-positive cases had frameshift mutations in the poly(A)₁₀ tract of the TGFβRII gene. This mutation was found only in the MSI-positive component in the 2 cases (cases 4 and 5) in which only 1 component exhibited MSI. The other 4 cases demonstrated homozygous or heteroclonal mutations (1 and 2 base deletions) in the poly(A)₈ tract of the hMSH3 gene; no mutation was detected in the poly(C)₈ tract of the hMSH6 gene in any of the MSI-positive cases. The profile of alterations in multiple targets was different between the 2 components in most of the cases (5/6). These findings suggest that mismatch repair deficiency in MSI-positive tumors causes multiple gene inactivations through frameshift mutations in short repetitive sequences in a heterogeneous way within a histologically heterogeneous tumor.     

Frameshift Mutations of the hMSH6 Gene in Human Leukemia Cell Lines

Defects in DNA mismatch repair mechanisms, including frameshift mutations of the hMSH6 and hMSH3 genes at their (C)₈ and (A)₈ tracks, respectively, have been shown to be associated with human malignancies. To clarify the possible involvement of these mutations in hematopoietic malignancies, we screened a total of forty-four human leukemia and lymphoma cell lines for mutations in the hMSH6 and hMSH3 genes, as well as in other genes required for DNA replication or repair, by polymerase chain reaction single-strand conformation polymorphism analysis and sequencing analysis. Frameshift mutations at the (C)₈ track of the hMSH6 gene were detected in two cell lines established from lymphoid leukemias. These two cell lines had no wild-type alleles, and both of them showed microsatellite instability. This is the first report that describes mutations and inactivation of the hMSH6 gene in hematological malignancies, suggesting that defects of the hMSH6 gene may be associated with development of hematological malignancies.     

A Novel Multidrug Resistance in Cultured Leukemia and Lymphoma Cells Detected by a Monoclonal Antibody to 85-kDa Protein, MRK20

A monoclonal antibody, MRK20, in F(ab')₂ form [MRK20-F(ab')₂], which reacts with 85-kDa membrane protein in a doxorubicin (ADM)-resistant subline (K562/ADM) of human myelogenous leukemia cell line, K562, was examined for reactivity with 41 cultured human leukemia and lymphoma cell lines. None of these cell lines had ever been exposed to any anticancer agent in vitro except K562/ ADM. The relative resistance index to various drugs was calculated by dividing the 50% growthinhibitory concentration (IC₅₀) of the test cell line by IC₅₀ of K562 (the negative control in the antibody experiment). MRK20-F(ab')₂ reacted with seven cell lines, KYO-1 derived from chronic myelogenous leukemia in blastic crisis (CMLbc), CMK from acute megakaryoblastic leukemia, HEL from erythroleukemia, P31/FUJ from acute monocytic leukemia, KOPM-28 from CMLbc, PL-21 from acute promyelocytic leukemia and K562/ADM, MRK20-F(ab')₂ did not react with 34 other cell lines. All seven MRK20-F(ab')₂-positive cell lines had relative resistance index values of 2 or more to anthracyclines (ADM, pyrarubicin, daunorubicin), mitoxantrone, etoposide, bleomycin, and pepleomycin. There was no distinct correlation between the reactivity to MRK20-F(ab')₂ and a higher relative resistance index than 2 to vinca alkaloids, actinomycin-D, cisplatin, 4-hydroperoxycyclophos-phamide, nimustine hydrochloride, methotrexate or cytarabine. These results indicate that MRK20-F(ab')₂ detects a novel multidrug resistance to anthracyclines, mitoxantrone, etoposide, bleomycin and pepleomycin in cultured human leukemia and lymphoma cells.     

Expression of Tumor-rejection Antigens in Gynecologic Cancers

We recently reported the four tumor-rejection antigens (SART1₂₅₉ SART2, SART3, and ART4) that possess tumor epitopes capable of inducing HLA-A2402-restricted cytotoxic T lymphocytes (CTLs) in cancer patients. This study investigated the expression of these tumor antigens in gynecologic cancers, including 33 ovarian cancers, 38 cervical cancers, and 40 endometrial cancers. SART1₂₅₉ antigen was detected in 56%, 35%, and 30% of ovarian, cervical and endometrial cancers, while SART2 antigen was detected in 46%, 66%, and 30% of these cancers, respectively. Both SART3 and ART4 antigens were detectable in the majority of these gynecologic cancers tested. In contrast, none of these antigens was detectable in any of the normal ovarian and uterine tissues tested. Peripheral blood mononuclear cells (PBMCs) of HLA-A24⁺ patients with gynecologic cancers were found to produce significant levels of interferon-γ in response to HLA-A24⁺ SART3⁺ gynecologic cancer cells after having been stimulated three times in vitro with either SART3_109–118 or SART3_315–323 peptide. These PBMCs lysed HLA-A24⁺ SART3⁺ gynecologic cancer cells, but not HLA-A24^- SART3⁺ gynecologic cancer cells or HLA-A24⁺ normal cells. Therefore, these four antigens and their peptides, including SART3 peptides, would be appropriate molecules for use in specific immunotherapy of HLA-A24⁺ gynecologic cancer patients.     

Efficient Gene Transduction by RGB-fiber Modified Recombinant Adenovirus into Dendritic Cells

Dendritic cells (DC) are important antigen-presenting cells in the development of an anti-tumor T cell response. To extend the range of current immuno/gene therapies, we tested luciferase-expressing RGD-adenovirus (Ad) (Ad5lucRGD)-mediated transduction into DC. Phenotypically characterized DC were generated from peripheral blood CD14⁺ cells by incubation with granulocyte-macrophage colony-stimulating factor, interleukin-4 and tumor necrosis factor a. On the 7th day of culture, the cells became mature DC with a CD1a⁺, CD11c⁺, CD80⁺, CD83⁺, CD86⁺, human leukocyte antigen (HLA)-DR⁺, CD14⁻ phenotype. The expression of (α,β₃ integrin was enhanced on day 3 and returned to the basal level on day 7. We then compared the transduction efficiency of an AdSlucRGD system to that using conventional Ad, in cells harvested on days 1, 3 and 7 of culture. Luciferase activity was negligible in AdCMVLuc, but remarkable in cells processed with Ad5lucRGD. Activity was maximal in cells that had been cultured for 3 days. Recombinant Ad5 fiber knob protein blocked AdCMVLuc- and Ad5lucRGD-mediated gene transduction by 90% and 20%, respectively. Surface markers and cytokine production were not affected by Ad5lucRGD-mediated transduction.     

Multidrug Resistance in Cultured Human Leukemia and Lymphoma Cell Lines Detected by a Monoclonal Antibody, MRK16

Forty cultured human leukemia and lymphoma cell lines never exposed to anticancer agents in culture, apart from doxorubicin (ADM)-resistant K562/ADM, were examined for reactivity with a monoclonal antibody, MRK16 in F(ab')₂ form [MRK16-F(ab')₂], which recognizes P-glycoprotein (P-gp). The relative resistance index to various drugs was calculated by dividing the 50% growth inhibitory concentration (IC₅₀) of the test cell line by IC₅₀ of K562, which was the negative control in the antibody experiment. MRK16-F(ab')₂ reacted with four cell lines, K562/ADM, KYO-1, HEL and CMK, which had relative resistance index values of 2 or more to vincristine (VCR), vindesine, vinblastine, ADM, daunorubicin, mitoxantrone (MIT), etoposide (VP-16) and actinomycin-D (ACT-D). The level of resistance to VCR and ADM in these cell lines decreased significantly in the presence of 10 μ M verapamil in vitro . Significant expression of mRNA of P-gp gene was also detected in K562/ADM, KYO-1 and HEL. MRK16-F(ab')₂ did not react with 36 other cell lines. Among them, three cell lines, PL-21, P31/FUJ and KOPM-28, had relative resistance index values of 2 or more to anthracyclines, MIT and VP-16, but not to vinca alkaloids or ACT-D. The level of ADM-resistance in these cell lines did not decrease significantly in the presence of 10 μ M verapamil. Five cell lines, ATL-1K, HL-60, KMOE-2, ML-1 and U266, had relative resistance index values of 2 or more to some of the drugs, but not to the others, and 19 other cell lines did not. These results indicate that the reactivity of MRK16-F(ab')₂ correlates with a relative resistance index of 2 or more to all these drugs in cultured human leukemia and lymphoma cell lines.     

Binding Sites of Droloxifene in the Cytosol of 7,12-Dimethylbenz[α]anthracene-induced Rat Mammary Tumor Cells

The binding sites, other than the estrogen receptor (ER), of the antiestrogens droloxifene (DROL, (E)-α-[ p [2-(dimethylamino)ethoxy]-phenyl]-α'-ethyl-3-stilbenol) and tamoxifen (TAM), and estradiol-17β (E₂) in the cytosol of 7,12-dimethylbenz[α]anthracene-induced rat mammary ER-positive tumor cells were studied using a high-performance liquid chromatography (HPLC) gel filtration assay. The cytosol was incubated with ³H-labeled drug with or without unlabeled drug, and separated by HPLC gel filtration. ³H-E₂ produced two major peaks of radioactivity at fractions No. 40 and No. 70. The peak at fraction No. 70 was identified as the ER in an ER-enzyme-immuno assay. This peak was dose-dependently inhibited by unlabeled DROL or TAM, DROL being a more potent inhibitor than TAM. The peak at fraction No. 40 was also inhibited by co-incubation with unlabeled DROL or TAM. ³H-DROL or ³H-TAM provided only one peak at fraction No. 43. This peak was thought to be an antiestrogen binding site (AEBS), because it was inhibited by unlabeled antiestrogen hut not by E₂. The results suggest that the antiestrogens DROL and TAM have a higher affinity for the AEBS than for the ER in the absence of E₂, while in the presence of E₂ both have an affinity for the ER and inhibit E₂ binding to the ER.     

Differential Alterations of Dihydrofolate Reductase Gene in Human Leukemia Cell Lines Made Resistant to Various Folate Analogues

In order to clarify a molecular mechanism of folate resistance in leukemia cells, we studied alterations of the dihydrofolate reductase (DHFR) gene in a human leukemia cell line, MOLT-3, and its sublines made resistant to methotrexate (MTX), trimetrexate (TMQ) and N¹⁰-propargyl-5,8-dideazafolic acid (CB3717), alone or in combination. Major alterations of the DHFR gene were examined by Southern analysis of high-molecular-weight DNA. The presence of a base change (T→C) at nucleotide position 91 of the DHFR gene, which is reported to be responsible for the reduced affinity of the enzyme for MTX in an MTX-resistant human colon carcinoma cell, was examined by allele-specific oligonucleotide hybridization. In a 10,000-fold MTX-resistant subline (MOLT-3/MTX_10,000), the normal allele of DHFR gene had been amplified. In contrast, a 200-fold TMQ-resistant subline (MOLT-3/TMQ₂₀₀) and a 30-fold CB3717-resistant subline selected from MOLT-3/TMQ₂₀₀ (MOLT-3/TMQ₂₀₀-CB-3717₃₀) were shown to have the mutant allele. Furthermore, the mutant allele had been amplified in a 500-fold MTX-resistant subline, which was established by the continuous exposure of the MOLT-3/TMQ₂₀₀ cells to stepwise increases of drug concentration and designated as MOLT-3/TMQ₂₀₀-MTX₅₀₀. On the other hand, a 40-fold-resistant subline to CB3717 alone (MOLT-3/CB3717₄₀) showed the normal allele without amplification. These data suggest that complex alterations of the DHFR gene are involved in the molecular mechanisms of folate resistance that can be differentially introduced into leukemia cells by exposure to various folate analogues, alone or in combination.     

Alterations of Integrin Expression in Human Lung Cancer

Integrins are cell-surface receptors which are involved in cell-matrix and/or cell-cell adhesion. They have been suggested to play a role in tumor invasion and metastasis. We examined the expression of various integrin subunits in normal and cancer cells of the lung using 33 human lung cancer cell lines as well as 6 lung cancer samples from which tumor cell lines could be established. This study clearly demonstrated that changes in the expression of certain integrins occur frequently in lung cancer, especially in small cell lung cancer. Loss of the α1 subunit of the β₁ integrin family appears to be the most prominent change, although loss of other integrin subunits such as α₂ or emergence of some integrin subunits such as α_v can also be observed. These results suggest that changes in integrin expression may contribute to the invasive and/or metastatic behavior of lung cancer.     

DNA Sequence of Immunoglobulin Heavy Chain Variable Region Gene in Thyroid Lymphoma

Patho–epidemiological studies have shown that thyroid lymphoma (TL) develops in thyroid affected by chronic lymphocytic thyroiditis (CLTH). CLTH is categorized as an organ–specific autoimmune disease, in which activated B–lymphocytes secrete a number of autoantibodies. Because antigenic stimulation might be involved in the pathogenesis of TL, the variable region in heavy chain ( V _H) genes was characterized in 13 cases with TL and 3 with CLTH. Clonal rearrangement of the V_H gene was found in 11 cases of TL, and cloning study with sequencing of complimen–tarity determining region (CDR) 3 revealed the presence of a major clone in 4. Three of the 4 cases used V_H3 gene, with the homologous germline gene of V3–30 in two cases and VH26 in one case. A biased usage of V_H3 and V_H4 genes with the homologous germline gene of VH26 in V_H3 gene was reported previously in cases with CLTH. A high level of somatic mutation (1–21%, average 12%) with non–random distribution of replacement and silent mutations was accumulated in all cases. The frequency of the occurrence of minor clones ranged from 29–44% per case, indicating the presence of on–going mutation. DNA sequencing of immunoglobulin V_H gene suggests that TL develops among activated lymphoid cells in CLTH at the germinal center stage under antigen selection     

Augmentation by Bispecific F(ab')2 Reactive with P-Glycoprotein and CD3 of Cytotoxicity of Human Effector Cells on P-Glycoprotein Positive Human Renal Cancer Cells

A bispecific F(ba')₂ was constructed that was composed of two Fab fragments, one derived from anti-CD3 monoclonal antibody (mAb) (OKT3) and the other from anti P-glycoprotein mAb (MRK 16). This bispecific F(ab')₂ enhanced the binding and cytotoxicity of human peripheral blood mononuclear cells (PBMCs) on P-glycoprotein-positive human kidney cancer cells (ADMHK/E). It had no effect on the cytotoxicity of PBMCs on P-glycoprotein-negative HK/E cells [long-term cultured HK/E (LCHK/E)]. Control F(ab')₂ composed of OKT3 or MRK16 alone did not influence the cytotoxicity of PBMCs on ADMHK/E cells. These findings suggest that the MRK16-OKT3 bispecific F(ab')₂ may be therapeutically beneficial in treatment of human multidrug-resistant cancers.     

Relationship between Cell-killing Efficiency and Number of Platinum Atoms Binding to DNA, RNA, and Protein Molecules in HeLa Cells Treated with cis-Diamine(glycolato)platinum(II)

HeLa S-3 cells were treated with ^195mPt-radiolabeled cis -diamine(glycolato)platinum(II) (254-S) under various conditions, and the relationship between the lethal effect and the numbers of Pt atoms binding to DNA, RNA, and proteins was examined. The mean lethal concentrations for the cells treated with 254-S at 37°C for 0.5, 1, 2, and 3 h were 67.1, 47.0, 26.8 and 8.1 μ M ₁, respectively. Using identically treated cells, we determined the numbers of Pt atoms combined with DNA, RNA, and protein molecules after fractionation of the cells. In this way, the D₀ values (D₀, the dose that causes an average of one lethal event per member of the population), expressed as the drug concentration, could he related to the number of Pt atoms combined with each fraction. The efficiency of the Pt atom in killing the cells, expressed as the reciprocal of the D₀ values, was then calculated for each fraction. The results suggested that DNA was the primary target for cell killing by 254-S. The target volumes for DNA were 3.96, 4.97, and 11.77 × 10⁴ nucleotides for 1-, 2-, and 3-h treated cells, respectively. In terms of the target volume, the cell-killing effects of 254-S were comparable to those of cis -diamine-dichloroplatinum(II) (CDDP), for which the target volumes under identical conditions were determined to be 5.17, 5.71, and 10.3 × 10⁴ nucleotides, respectively, while in terms of the mean lethal dose (D₀), the cell-killing effects of 254-S were lower than those of CDDP by a factor of 5.1 (47.0/9.3), 4.0 (26.8/6.7), or 2.5 (8.1/3.2) for 1-, 2-, or 3-h treatment, respectively.     

The Effects of Boron Neutron Capture Therapy on Liver Tumors and Normal Hepatocytes in Mice

To explore the feasibility of employing boron neutron capture therapy (BNCT) to treat liver tumors, the effects of BNCT were investigated by using liver tumor models and normal hepatocytes in mice. Liver tumor models in C3H mice were developed by intrasplenic injection of SCCVII tumor cells. After borocaptate sodium (BSH) and boronophenylalanine (BPA) administration, ¹⁰B concentrations were measured in tumors and liver and the liver was irradiated with thermal neutrons. The effects of BNCT on the tumor and normal hepatocytes were studied by using colony formation assay and micronucleus assay, respectively. To compare the effects of BSH-BNCT and BPA-BNCT, the compound biological effectiveness (CBE) factor was determined. The CBE factors for BSH on the tumor were 4.22 and 2.29 using D ₁₀ and D ₀ as endpoints, respectively. Those for BPA were 9.94 and 5.64. In the case of hepatocytes, the CBE factors for BSH and BPA were 0.94 and 4.25, respectively. Tumor-to-liver ratios of boron concentration following BSH and BPA administration were 0.3 and 2.8, respectively. Considering the accumulation ratios of ¹⁰B, the therapeutic gain factors for BSH and BPA were 0.7-1.3 and 3.8-6.6, respectively. Therefore, it may be feasible to treat liver tumors with BPA-BNCT.