Biomedical subjects

A F Kirkin

Publications and source records attributed to A F Kirkin.

At least 19 recordsLinked to original sources

PubMed2006

Vaccination with melanoma lysate-pulsed dendritic cells, of patients with advanced colorectal carcinoma: report from a phase I study.

Immune therapy have shown new and exciting perspectives for cancer treatment. Aim of our study was to evaluate toxicity and possible adverse effects from vaccination of patients with advanced colorectal cancer with autologous dendritic cells (DC) pulsed with lysate from a newly developed melanoma cell line, DDM-1.13. Six patients were enrolled in the phase I trial. Autologous DCs were generated in vitro from peripheral blood monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were pulsed with melanoma cell lysate from a cloned and selected melanoma cell line enriched in expression of MAGE-A antigens and deficient in expression of melanoma differentiation antigens: tyrosinase, MART-1 and gp100. Vaccinations were administered intradermally on the proximal thigh with a total of five given vaccines at 2 weeks intervals. Each vaccine contained 3-5 x 10(6) DCs. Five of the six patients received all five vaccines. The treatment was well tolerated in all patients without any observed vaccine-correlated adverse effects. Treatment with this DC-based cancer vaccine proved safe and non-toxic.

Adult↗

PubMed2001

Methylation-dependent silencing of the reduced folate carrier gene in inherently methotrexate-resistant human breast cancer cells.

The molecular basis of methotrexate resistance was studied in human MDA-MB-231 breast cancer cells, which are inherently defective in methotrexate uptake and lack expression of the reduced folate carrier (RFC). Transfection of MDA-MB-231 cells with RFC cDNA restored methotrexate uptake and increased methotrexate sensitivity by approximately 50-fold. A CpG island in the promoter region of RFC was found to be methylated in MDA-MB-231 cells, but was unmethylated in RFC expressing, methotrexate-sensitive MCF-7 breast cancer cells. Chromatin immunoprecipitation with antibodies against acetylated histones H3 and H4 showed that the RFC promoter was enriched for acetylated histones on expressed, unmethylated alleles only. Treatment of MDA-MB-231 cells with 5-aza-2'-deoxycytidine restored RFC expression but also led to increased methotrexate efflux and did not reverse methotrexate resistance. This suggests that 5-aza-2'-deoxycytidine up-regulates both methotrexate uptake and some methotrexate-resistance mechanism(s). Reverse transcription-polymerase chain reaction analysis showed increased expression levels of several ATP-dependent efflux pumps in response to 5-aza-2'-deoxycytidine treatment, including P-glycoprotein and members of the multidrug resistance-associated protein family. Up-regulation of P-glycoprotein in response to 5-aza-2'-deoxycytidine was associated with demethylation of a CpG island in the MDR1 promoter, whereas the mechanism(s) for 5-aza-2'-deoxycytidine-induced up-regulation of multidrug resistance-associated proteins is probably indirect. Dipyridamole inhibited methotrexate efflux and reversed methotrexate resistance in 5-aza-2'-deoxycytidine-treated MDA-MB-231 cells.

Acetylation↗

PubMed2000

Aberrant p27Kip1 promoter methylation in malignant melanoma.

p27Kip1 is a regulator of the mammalian cell cycle and a putative tumor suppressor. Distinct altered patterns of p27Kip1 protein expression are found in a variety of human carcinomas, and p27Kip1 expression levels usually correlate directly with disease-free survival. The mechanism(s) by which p27Kip1 expression is reduced or lost during tumorigenesis remains unclear. Specific alterations of the p27Kip1 gene, including mutations and homozygous deletions, are exceedingly rare in human cancers. We have used methylation-specific PCR and bisulfite genomic sequencing to examine the methylation status of p27Kip1 in 61 primary and metastatic tumors and 35 cell lines from patients with malignant melanoma. Dense methylation of a CpG island in the promoter region of p27Kip1 was detected in four of 45 metastatic tumors (9%) and three of the cell lines (9%), including two cell lines established from two different metastases from the same patient. Examination of a naturally occurring, allele-specific sequence variant demonstrated that p27Kip1 methylation is associated with transcriptional silencing in situ. Cell lines with p27Kip1 methylation showed retention of the wild-type allele and detectable p27Kip1 protein whose abundance was reduced compared with normal melanocytes. Collectively, our data suggest that DNA methylation may be a cause of monoallelic p27Kip1 silencing in malignant melanoma, which would support a role for p27Kip1 haploinsufficiency in human cancer.

Amino Acid Substitution↗

PubMed2000

Recombinant human single-chain MHC-peptide complexes made from E. coli By in vitro refolding: functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens.

Soluble recombinant MHC-peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies. In this study, soluble recombinant single-chain human MHC (scMHC)-peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA-A2 in the presence of tumor-associated or viral peptides. The scMHC molecule was composed of beta2-microglobulin connected to the first three domains of the HLA-A2 heavy chain through a 15-amino acid flexible linker. Highly purified scMHC-peptide complexes were obtained in high yield using several peptides derived from the melanoma antigens gp100 and MART-1 or a viral peptide derived from HTLV-1. The scMHC complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA-A2-restricted peptides. We also generated scMHC-peptide tetramers, which were biologically functional; they induced a peptide-specific CTL clone to be activated and secrete IFN-gamma, and were able to stain specifically CTL lines. Such recombinant soluble scMHC-peptide complexes and tetramers should prove of great value for characterization of immune responses involving CTL, for visualization of antigen-specific immune responses, for in vitro primary CTL induction, and for peptide binding assays and structural studies.

Antigens, Neoplasm↗

PubMed2000

Tumor infiltrating lymphocytes in melanoma comprise high numbers of T-cell clonotypes that are lost during in vitro culture.

Melanoma is generally accepted as being an antigenic tumor capable of eliciting T-cell responses that, however, in most cases are inadequate to control tumor growth. Tumor-infiltrating lymphocytes (TIL) in melanoma lesions comprise clonotypic T cells, indicating the in situ recognition of melanoma-associated peptide epitopes. Cultured TIL have been studied in order to unveil characteristics of TIL and the interactions of TIL and melanoma cells. Whether in vitro cultured TIL mirrors the in situ situation has, however, been questioned. In the present study we have taken advantage of T-cell receptor clonotype mapping methodology to conduct a full and detailed analysis of the T-cell clonotypes in melanoma lesions and in corresponding lines of TIL established in vitro. All melanoma lesions and the corresponding TIL cultures comprised high numbers of T-cell clonotypes, typically in the range of 40 to more than 60. The subsequent comparison of T-cell clonotypes present in the original lesions and in the corresponding T-cell lines established in vitro demonstrated that a very limited number of the T-cell clonotypes established in vitro are identical to the T-cell clonotypes expanded in situ. These results demonstrate that in situ T-cell clonotypes in melanoma are not readily expanded in vitro and that the majority of T-cell clonotypes present in cultured TIL are not present in situ.

Cell Count↗

PubMed2000

Cytotoxic T-lymphocyte clones, established by stimulation with the HLA-A2 binding p5365-73 wild type peptide loaded on dendritic cells In vitro, specifically recognize and lyse HLA-A2 tumour cells overexpressing the p53 protein.

Mutations in the tumour suppressor gene p53 are among the most frequent genetic alterations in human malignancies, often associated with an accumulation of the p53 protein in the cytoplasm. We have generated a number of cytotoxic T lymphocyte (CTL) clones that specifically recognize the HLA-A*0201 p53 wild type peptide RMPEAAPPV [65-73], designated R9V, by the in vitro stimulation of CD8 enriched peripheral blood lymphocytes from a healthy HLA-A*0201 donor using peptide loaded autologous dendritic cells. A total of 22 CTL clones were generated from the same bulk culture and demonstrated to carry identical T-cell receptors. The CTL clone, 2D9, was shown to specifically lyse the HLA-A*0201+ squamous carcinoma cell line SCC9 and the breast cancer cell line MDA-MB-468. Our data demonstrate that human peripheral blood lymphocytes from normal healthy individuals comprise T cells capable of recognizing p53 derived wild type (self) peptides. Furthermore, the capacity of R9V specific T cell clones to exert HLA restricted cytotoxicity, argues that the R9V peptide is naturally presented on certain cancer cells. This supports the view that p53 derived wild type peptides might serve as candidate target antigens for the immunotherapeutic treatment of cancer.

Cell Line, Transformed↗

PubMed1999

In situ T cell responses against melanoma comprise high numbers of locally expanded T cell clonotypes.

It is well established that melanoma cells express Ags that are recognized by autologous T cells in vitro. Tumor-infiltrating lymphocytes in situ comprise clonotypic T cells, suggesting that their expansion is driven by Ag stimulation. Still, little is known about the detailed characteristics of the in situ T cell response. In the present study, we scrutinized this response by analyzing multiple metastatic lesions for the presence of clonotypic T cells. This approach was chosen to distinguish whether the clonal T cell expansion occurs as a systemic or localized phenomenon. TCR clonotype mapping of six s.c. metastases from two patients revealed the presence of multiple (from 40 to >60) clonotypic T cells in all lesions. Clonotypic T cells were present in TCR beta-variable regions expressed both at high and low levels. Comparison of the T cell clonotypes present in different lesions from individual patients demonstrated that, in general, clonotypes were exclusively detected in a single lesion. Hence, anti-melanoma T cell responses are much more heterogeneous than previously anticipated and accommodate a predominance of strictly localized T cell clonotypes.

Antigens, Neoplasm↗

PubMed1999

Somatic mutation of the Peutz-Jeghers syndrome gene, LKB1/STK11, in malignant melanoma.

Mutations in LKB1/STK11, a gene mapping to chromosome 19p13.3 and encoding a widely expressed serine/threonine kinase, were recently identified as the cause of Peutz-Jeghers syndrome. Despite the hamartomatous polyps and increased cancer risk associated with this syndrome, somatic alterations in LKB1/STK11 have not been identified in human tumours. Prompted by another feature of the syndrome, lentigines of the lips and oral mucosa, we evaluated the status of LKB1/STK11 expression, deletion, and mutation in cell lines and tumour samples from 35 patients with sporadic malignant melanoma. Two somatic mutations were identified, a nonsense mutation (Glu170Stop) causing exon skipping and intron retention, and a missense mutation (Asp194Tyr) affecting an invariant residue in the catalytic subunit of LKB1/STK11. Our data suggest that LKB1/STK11 may contribute to tumorigenesis in a small fraction of malignant melanomas.

AMP-Activated Protein Kinase Kinases↗

PubMed1999

Establishment of gp100 and MART-1/Melan-A-specific cytotoxic T lymphocyte clones using in vitro immunization against preselected highly immunogenic melanoma cell clones.

The induction of an in vitro T cell response against tumour-associated antigens with subsequent expansion of the individual cytotoxic T lymphocyte (CTL) clones still is not routine and the only tumour-associated antigen that has been found to easily induce the establishment of CTL clones is the MART-1/Melan-A antigen. In this paper, we describe a new approach for in vitro immunization based on the use of preselected melanoma cell clones. The human melanoma cell subline FM3.P was cloned and the immunological properties of individual clones were compared. Melanoma cell clone FM3.29, having a high level of expression of melanoma differentiation antigens, as well as high levels of the HLA class I and class II antigens and adhesion molecules, was used for the establishment of a CTL line that was subsequently cloned. For optimization of the conditions of growth of established CTL clones, a particular melanoma subline FM3.D/40 was selected for supporting the proliferation of CTL clones. The majority of the established CTL clones recognized the melanoma-associated differentiation antigens gp100 and MART-1/Melan-A. Epitope analysis indicated that two different epitopes derived from gp100 (154-162 and 280-288) and a single epitope from MART-1/Melan-A (27 35) were recognized by these CTL clones. The gp100-specific CTL clones were found to be significantly more sensitive to the culture conditions than the MART-1/Melan-A-specific CTL clones. In addition, the presence of excess peptide in the culture medium induced autokilling of the gp100-specific, but not the MART-1/Melan-A-specific CTL clones. Taken together, these results demonstrate that, by careful preselection of melanoma cell lines and clones both for the induction of CTL line from patients' peripheral blood lymphocytes and subsequent cloning, it is possible to obtain a large number of stable CTL clones even against such an inherently "difficult" differentiation antigen as gp100.

Antigens, Neoplasm↗

PubMed1999

Expression of basic fibroblast growth factor and vascular endothelial growth factor in primary and metastatic melanoma from the same patients.

Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are both recognized as stimulators of migration and angiogenesis during the progression of melanoma. However, the timepoints during tumour progression at which the expression of these angiogenic factors is most essential is still controversial. Using immunohistochemical analyses, melanoma cells were found to express bFGF in 18 out of 19 primary tumours and in 13 out of 20 metastases. Eleven of the 19 primary tumours and 15 of the 20 metastases were found to contain VEGF-positive melanoma cells; five of the 19 patients showed no VEGF-expressing melanoma cells at all. This indicates that VEGF expression may be a later event in the progression of melanoma than bFGF expression. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of the melanoma cell lines showed that all cell lines were positive for both bFGF and VEGF mRNA. CD31-positive endothelial cells were primarily seen in the metastases (17 out of 20). Only four of the primary tumours contained CD31-positive cells, but these tumours expressed bFGF as well as VEGF, indicating that both angiogenic factors may be important for the formation of vessels in tumours.

Adult↗

PubMed1998

In vivo eradication of an established human melanoma by an in vitro generated autologous cytotoxic T cell clone: a SCID mouse model.

Tumor-specific T cells may be induced in vitro and in vivo. A tumor, however, is able to avoid recognition by T cells by various mechanisms, and it has therefore been difficult to use these cells for the treatment of cancer. To investigate these mechanisms, it would be desirable to identify a suitable in vivo model system to avoid the ethical considerations that are obviously limiting factors for studies in humans. In addition, tumor antigens, although recognized, may not always function as rejection antigens, thus, the establishment of an in vivo model is crucial for preclinical studies to allow the characterization of effective rejection antigens. We show here that the immunodeficient scid mouse is an excellent model system. Using this system, we demonstrate that an already established human melanoma tumor is eradicated by an in vitro generated autologous cytotoxic T cell clone.

Animals↗

PubMed1998

Ecto-ATP diphosphohydrolase/CD39 is overexpressed in differentiated human melanomas.

Ecto-ATPase activities of melanocytes and human melanoma cell lines differing in the stage of progression were compared. A dramatic increase in ecto-ATPase activity above the level of normal melanocytes was demonstrated in the differentiated melanomas and was followed by a gradual decrease with tumor progression. The characteristics of this enzymatic activity were consistent with CD39/ecto-ATP diphosphohydrolase (ATPDase) which was found to be the major ecto-ATP-hydrolysing enzyme in melanomas. Indeed, the expression of CD39 and the level of CD39 mRNA followed a similar pattern. Since CD39 is known to regulate homotypic adhesion and, supposedly, affects the disaggregation step, we suggest that overexpression of CD39 may enable tumor cells to reduce contacts with T-lymphocytes and escape from immunological recognition.

Adenosine Triphosphatases↗

PubMed1998

Generation and characterization of gp100 peptide-specific NK-T cell clones.

MHC-restricted cytotoxic T lymphocytes (CTLs) specific for antigens expressed by malignant cells are important components of immune responses against human cancer. Peripheral blood monocytes of HLA-A2+ healthy donors were used to induce dendritic cells (DCs) by granulocyte-macrophage colony-stimulating factor and interleukin-4 and loaded with a gp100 peptide (YLEPGPVTA). By applying these peptide-loaded DCs, a CTL line that displayed high cytotoxic reactivity with peptide-loaded target cells was generated. A total of 11 gp100 peptide-specific CTL clones were generated from this cell line. Several of these CTL clones were studied in detail. Of particular interest was clone CTL-45, which, contrary to the parental cell line, displayed strong NK activity and, by flow-cytometric analysis, revealed a CD3+, TCR BV17, CD8+ and CD56+ phenotype. This clone was strictly peptide-specific and effectively killed a panel of melanoma cells expressing HLA-A2 and gp100. Tumor-specific T cells with this kind of dual function are potentially of great clinical importance as they have a backup mechanism that may go into action when tumor cells escape specific killing by losing their HLA-class I molecules.

Amino Acid Sequence↗

PubMed1998

Melanoma-associated antigens recognized by cytotoxic T lymphocytes.

During the last 7 years significant progress has been made in the identification of melanoma-associated antigens recognized by cytotoxic T lymphocytes (CTL). These antigens belong to three main groups: cancer/testis-specific antigens (MAGE, BAGE, GAGE, PRAME and NY-ESO-1), melanocyte differentiation antigens (tyrosinase, Melan-A/MART-1, gp100, TRP-1 and TRP-2), and mutated or aberrantly expressed antigens (MUM-1, CDK4, beta-catenin, gp100-in4, p15 and N-acetylglucosaminyltransferase V). In this review we have summarized the available data concerning the characterization of melanoma-associated antigens, focusing on their immunogenic and protective properties. The development of a strong immune response to differentiation antigens is limited by the existence of tolerance to these "self"-antigens, permitting the involvement of only T cells with low affinity T-cell receptors. Among the melanoma differentiation antigens, only gp100 has been shown to be a tumor regression antigen. The cancer/testis-specific antigens such as MAGE and PRAME should potentially be highly immunogenic antigens. They contain several potential HLA class I binding epitopes and are present only in the testes, which are not accessible to the cells of the immune system owing to the lack of direct contact with the immune cells and the lack of HLA class I expression on the surface of germ cells. But only two patients have been found who responded to these antigens in vivo, indicating their genuinely low immunogenicity. A comparison of the predicted secondary structures of these two groups of antigens (cancer/testis-specific and differentiation antigens) revealed enrichment of long alpha-helical stretches in the cancer/testis-specific antigens. We hypothesize that such highly organized stable structures could, first, reduce denaturation of the protein and, thus, ubiquitinylation as a degradation signal, and, second, diminish the efficiency of the protein unfolding - a necessary step in the proteolytic cleavage by proteasomes. High structural stability could therefore be responsible for the low immunogenicity of these proteins. In this case, modifications decreasing the stability of these proteins might be a means of improving the immune response to these potentially therapeutically useful antigens.

Amino Acid Sequence↗

PubMed1998

The immunogenic properties of melanoma-associated antigens recognized by cytotoxic T lymphocytes.

During the last 6 years significant progress has been achieved in the identification of melanoma-associated antigens recognized by cytotoxic T lymphocytes. These antigens belong the three main groups: tumor-associated testis-specific antigens (MAGE, BAGE, GAGE and PRAME), melanocyte differentiation antigens (tyrosinase, Melan-A/MART-1, gp100, TRP-1 and TRP-2) and mutated or aberrantly expressed antigens (MUM-1, CDK4, beta-catenin, gp100-in4, p15 and N-acetylglucosaminyltransferase V). In this review, we have summarized the available data concerning the characterization of melanoma-associated antigens with focus on their immunogenic and protective properties. The development of a strong immune response against differentiation antigens is limited by the existence of tolerance against these 'self' antigens, permitting the involvement of only T cells with low affinity T cell receptors. Among the melanoma differentiation antigens, only gp100 has been shown to be a tumor regression antigen. The testis-specific antigens such as MAGE and PRAME should potentially be highly immunogenic antigens. They contain several potential HLA class I binding epitopes and are present only in the testes which are not accessible to the cells of the immune system due to the lack of direct contact with the immune cells and the lack of HLA class I expression on the surface of germ cells. But only 2 patients have been found who responded to these antigens in vivo, indicating their genuinely low immunogenicity. A comparison of the predicted secondary structures of these two groups of antigens (testis-specific and differentiation antigens) revealed enrichment of long alpha-helical stretches in the testis-specific antigens. We hypothesize that such highly organized structures could diminish the efficiency of the protein unfolding--a necessary step in the proteolytic cleavage by proteasomes--and, therefore, could be responsible for the low immunogenicity of these proteins. In this case, modifications decreasing the stability of these proteins might be a means to improve the immune response against these potentially therapeutically useful antigens.

Amino Acid Sequence↗

PubMed1997

Complete scanning of the CDK4 gene by denaturing gradient gel electrophoresis: a novel missense mutation but low overall frequency of mutations in sporadic metastatic malignant melanoma.

The cyclin-dependent kinase 4 (CDK4) is a key component in regulation of the mammalian cell cycle. The recent discovery of a common missense mutation (Arg24Cys) in both sporadic and familial forms of malignant melanoma strongly supports the candidacy of CDK4 as a proto-oncogene. To study further the role of CDK4 in melanoma pathogenesis, we have established a method based on polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) to scan the CDK4 gene for point mutations. By analyzing the entire coding sequence of the CDK4 gene in 56 sporadic metastatic malignant melanomas, we identified a novel missense mutation, Asn41Ser. This mutation was also found in the germline of the patient who had no family history of melanoma. Analysis of a tumor-derived cell line demonstrated equal expression of the mutant and wild-type CDK4 alleles, together with lack of functional p16. Our findings suggest that an oncogenic mechanism of the CDK4-Asn41Ser variant would be different from the CDK4-Arg24Cys variant. Altogether, our data demonstrate that point mutation of CDK4 is a rare event in melanoma pathogenesis.

Asparagine↗

PubMed1997

Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanoma.

The MMAC1/PTEN gene, located at 10q23.3, is a candidate tumor suppressor commonly mutated in glioma. We have studied the pattern of deletion, mutation, and expression of MMAC1/PTEN in 35 unrelated melanoma cell lines. Nine (26%) of the cell lines showed partial or complete homozygous deletion of the MMAC1/PTEN gene, and another six (17%) harbored a mutation in combination with loss of the second allele. Mutations could also be demonstrated in uncultured tumor specimens from which the cell lines had been established, and cell lines derived from two different metastases from one individual carried the same missense mutation. Collectively, these findings suggest that disruption of MMAC1/PTEN by allelic loss or mutation may contribute to the pathogenesis or neoplastic evolution in a large proportion of malignant melanomas.

Base Sequence↗

PubMed1997

Isolation of novel HLA-DR restricted potential tumor-associated antigens from the melanoma cell line FM3.

Endogenous peptides bound to the constitutively expressed MHC class II molecules HLA-DR and HLA-DQ of the melanoma cell line FM3 were examined. By a combination of analytical methods (narrow bore and capillary reversed-phase high-performance liquid chromatography with subsequent spotting on polyvinylidene difluoride membranes, matrix-assisted laser desorption ionization mass spectrometry, and Edman microsequencing), we were able to isolate and identify a panel of HLA-DR4/2 (HLA-DRB1*0401/0201/DRB5*0101)-associated self-peptides from the melanoma cell line FM3. Among ubiquitously HLA-DR-associated peptides such as peptides from the class II-associated invariant chain peptide region of the invariant chain, HLA-class I, the transferrin receptor, and the IFN-gamma receptor, we identified several potential tumor-associated antigens stemming from the MHC class I-restricted tumor antigen gp100, the Ca2(+)-binding protein annexin II, and proteins from the hsp70 family. Chinese hamster ovary cells cotransfected with HLA-DRA, DRB1*0401, and CD80 genes were shown specifically to prime T lymphocytes from HLA-DRB1*0401 donors to the annexin II and gp100 peptides. These results demonstrate that MHC class II molecules expressed by melanoma cells potentially present a variety of novel antigens to the immune system, some of which could be exploited for immunotherapy.

Animals↗