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Biomedical subjects

C Huber

Publications and source records attributed to C Huber.

At least 145 records · Page 8Linked to original sources
PubMed1996

Interferon alpha-2c therapy of patients with chronic myelogenous leukemia: long-term results of a multicenter phase-II study. Austrian Biological Response Modifier (BRM) Study Group.

In a prospective multicenter phase-II trial 80 patients with Philadelphia (Ph)-positive chronic myelogenous leukemia (CML) were treated with recombinant interferon (IFN) alpha-2c, administered subcutaneously at an absolute dose of 3.5 megaunits (MU)/day. Complete hematological remission was achieved in 29 (39%) and partial hematological remission in 26 (35%) of the 74 patients evaluable for response. Major cytogenetic responses were observed in ten (13%) and minor cytogenetic responses in 11 patients (15%). Median duration of cytogenetic response was 33 months (range, 2-90); relapses were seen in all of the 11 patients with minor and in three of the ten patients with major cytogenetic responses. Median survival estimates for pretreated (n = 19) and untreated (n = 58) patients were 51 months (95% confidence interval [CI], 30-72) and 77 months (95% CI, 43-111), and the survival probabilities at 5 years were 45% and 54% for the two groups, respectively. Hematological response after 3 months of treatment demonstrated a clear-cut discriminative capacity with 5-year survival probabilities of 100%, 67% and 24% for patients achieving CHR (n = 6), PHR (n = 34), and less than PHR (n = 35), respectively. Landmark analysis at 12, 18, and 24 months after start of IFN therapy and an analysis treating time to cytogenetic response as a time-dependent covariate showed that cytogenetic response was associated with longer survival. The impact of a low-dose IFN regimen on survival in CML patients is unclear and requires further clarification by randomized clinical trials. Early hematological and cytogenetic response to IFN-alpha treatment identifies patients with a favorable long-term prognosis.

Adolescent↗
PubMed1996

Regulation of immunomodulatory functions by granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in vivo.

The present study was designed to investigate in vivo immunomodulatory properties of hematopoietic growth factors. The influence on the activation of cytokine synthesis and on the expression of surface antigens associated with cellular activation of G-CSF or GM-CSF was investigated in cancer patients receiving these factors. One single dose of growth factor was administered to patients with bladder cancer (G-CSF group) or small cell lung cancer (GM-CSF group) before chemotherapy. After cytoreductive chemotherapy patients received supportive therapy with G-CSF or GM-CSF. Peripheral blood mononuclear cells and plasma samples were obtained for flow cytometry, Northern blot analysis, and assessment of cytokine protein levels after single-dose as well as after continuous cytokine administration. Our results demonstrate differences in the induction of biological activities by GM-CSF and G-CSF in vivo which correlate well with in vitro findings. Among mature hematopoietic cells the effect of G-CSF is restricted to the granulocyte lineage. With GM-CSF moderate but unequivocal modulation of monocyte function was observed. On peripheral blood monocytes expression of MHC class-II molecules and CD44 was markedly stimulated. After one single dose of GM-CSF, plasma levels of sCD25 and IL-1RA were significantly induced (p < 0.0001, p = 0.032, respectively) and a trend to increased IL-8 levels was observed. The changes in plasma proteins were not correlated with shifts of mRNA expression for IL-8 and IL-1RA. T-cell activation was not observed with either cytokine. These results suggest that immunomodulatory features are differentially regulated by G-CSF and GM-CSF. The clinical relevance of a selective use of both hematopoietic growth factors in various disease settings remains to be determined.

Adjuvants, Immunologic↗
PubMed1996

Radical cystectomy with or without adjuvant polychemotherapy for non-organ-confined transitional cell carcinoma of the urinary bladder: prognostic impact of lymph node involvement.

OBJECTIVES: To analyze the effectiveness of adjuvant polychemotherapy after radical cystectomy for non-organ-confined transitional cell bladder cancer (Stages pT3b, pT4a, and/or pN1 or pN2). METHODS: Of 166 consecutive patients undergoing cystectomy at two institutions from 1987 to 1993, 80 received adjuvant polychemotherapy with methotrexate, vinblastine, and cisplatin plus doxorubicin (MVAC) or epirubicin (MVEC), whereas 86 had cystectomy only. The patients were evaluated for relapse-free survival and length of progression-free interval on the basis of follow-up data obtained in 1995 and 1996. RESULTS: Kaplan-Meier analysis revealed a significantly higher progression-free rate for patients after adjuvant chemotherapy (P = 0.0002, log-rank test). With and without adjuvant chemotherapy, prognosis declined in a stepwise manner, depending on the extent of lymph node involvement. Nevertheless, the superior prognosis of the chemotherapy group could be demonstrated at each lymph node stage. Of the 166 patients, 49 had initially entered a prospective trial comparing adjuvant with no adjuvant treatment. That study was discontinued in December 1990 after an interim analysis revealed a significant prognostic advantage in favor of the 26 patients randomized to receive chemotherapy compared with the 23 control patients. Current follow-up data continue to demonstrate a significant improvement in progression-free survival in favor of patients randomized to receive adjuvant chemotherapy (P = 0.0040). The follow-up period of patients living free of disease ranges from 58 to 96 months. CONCLUSIONS: Adjuvant chemotherapy with MVAC/MVEC leads to significant prolongation of relapse-free survival and improvement of the definitive cure rate after radical cystectomy for locally advanced transitional cell carcinoma of the urinary bladder.

Adult↗
PubMed1996

CD4 monoclonal antibody VIT4 in human alloimmune response in vitro and in vivo.

In the present report the immunosuppressive effects of the murine anti-human CD4 monoclonal antibody (mAb) VIT4 on human alloimmune response in vitro were analyzed. Moreover, the antibody was tested for its activity to prolong allograft survival in seven patients with steroid-refractory allograft rejection. VIT4 inhibited the proliferative response to alloantigens in the mixed lymphocyte reaction (MLR) in a dose-dependent manner. At concentrations of 1 and 10 micrograms/ml VIT4 blocked MLR by 55 +/- 11% and 77 +/- 1%, respectively. Also alloantigen-specific proliferation of in vitro- generated memory T cells was dose-dependently reduced to 23 +/- 1% at a VIT4 concentration of 100 micrograms/ml. Furthermore, at the same dose level VIT4 blocked proliferation of antigen-specific short-term alloreactive CD4+ cell lines and significantly inhibited the in vitro generation of cytotoxic T lymphocytes (CTL). In a pilot study VIT4 (5 mg/d i.v.) was administered to 7 patients with steroid-refractory allograft rejection for 14 days. In 4 of 7 patients graft function transiently improved and graft survival in all patients was prolonged to a mean of 694 days (range 128-2163) from the beginning of the VIT4 treatment. In the light of our in vitro results and the preliminary clinical data, further clinical trials using higher antibody doses are greatly warranted to assess the efficacy of anti-CD4 mAb VIT4 in the treatment of allograft rejection.

Animals↗
PubMed1996

Induction of interferon regulatory factors 2'-5' oligoadenylate synthetase, P68 kinase and RNase L in chronic myelogenous leukaemia cells and its relationship to clinical responsiveness.

The genes crucially determining the therapeutic response of chronic myelogenous leukaemia (CML) to interferon-alpha (IFN-alpha) are unknown. Recently, two independent IFN-alpha signalling pathways were identified: the classic pathway mediates induction of 2'-5' oligoadenylate synthetase (2-5 OAS), p68 kinase and IFN regulatory factor-2 (IRF-2), whereas the alternate pathway leads to activation of IFN regulatory factor-1 (IRF-1). We investigated whether deficient or imbalanced expression of components of these two pathways is associated with resistance of CML cells to antiproliferative action of IFN alpha/beta. Constitutive and IFN-induced transcript levels of IFN-dependent genes in mononuclear cells, granulocytes, monocytes, lymphocytes and CD34+ cells of chronic-phase CML and blast crisis patients were assessed by Northern blot techniques and were correlated with subsequent clinical responses to IFN therapy. Our results demonstrated that IFN-alpha or -beta treatment in vitro and in vivo leads to an enhanced expression of IRF-1, IRF-2. RNase L, p68 and 2-5 OAS which was independent of the degree of cellular differentiation and clonal evolution of CML. Neither the magnitude of induction of these genes nor the IRF-1/IRF-2 mRNA balance differed between chronic-phase CML patients responding or failing IFN-alpha therapy. These results indicate that failure of IFN-alpha treatment is not due to defects in mRNA induction of the above-mentioned candidate genes for the direct antiproliferative response to IFN type I.

2',5'-Oligoadenylate Synthetase↗
PubMed1996

Primary proliferating immature myeloid cells from CML patients are not resistant to induction of apoptosis by DNA damage and growth factor withdrawal.

Induction of apoptosis by growth factor deprivation or gamma-irradiation-induced DNA damage was directly studied in proliferating primary haemopoietic cells derived from CD34-positive cells of 13 CML patients and 12 normal controls. CD34-positive cells were cultured in the presence of appropriate concentrations of SCF and G-CSF for 5-7 d. After gamma irradiation with 500 rad or growth factor deprivation, the fraction of apoptotic cells was assessed by two independent methods applying either measurement of cells incorporating FITC-labelled dUTP by terminal transferase or assessment of the fraction of cells with a less than 2N DNA content in flow cytometry. Proliferating CML cells were not resistant to the induction of apoptosis either after gamma irradiation or subsequent to growth factor deprivation. A similar fraction of normal and CML cells underwent apoptosis 48 h after withdrawal of growth factors. CML cells displayed an increased susceptibility to induction of apoptosis after DNA damage. A significantly higher proportion of apoptotic cells were detected in samples derived from CML patients after irradiation with 0.5 Gy. These results, in conjunction with conflicting observations by other investigators, on the induction of apoptosis by gamma irradiation in various bcr-abl positive cells, suggest that bcr-abl-dependent effects on apoptosis strongly depend on the cells used. Our observations in CML cells derived exclusively from newly diagnosed CML patients demonstrate that bcr-abl expression per se is not sufficient to induce resistance to apoptosis.

Apoptosis↗
PubMed1996

Recognition of human renal cell carcinoma and melanoma by HLA-A2-restricted cytotoxic T lymphocytes is mediated by shared peptide epitopes and up-regulated by interferon-gamma.

Cytotoxic T lymphocytes (CTL) have previously been isolated from peripheral blood of patients with renal cell carcinoma (RCC). The CD8-positive CTL line MZ1257-CTL-5 (CTL-5) has been shown to lyse autologous cultured RCC cells in an HLA-A2 restricted fashion. Allogeneic, HLA-A2-matched RCC and melanoma cell lines were also lysed by CTL-5, suggesting that melanoma and renal cancer share antigenic determinants. The aim of the study was to determine whether RCC and melanoma share peptide epitopes that are recognized by CTL-5 in the context of HLA-A2 molecules. Peptides were acideulated from various cell lines, separated by reversed phase high performance liquid chromatography (RP-HPLC), and assessed for their ability to reconstitute the CTL-5-defined epitope by pulsing the peptides on HLA-A2 positive antigen-processing mutant cell line CEM x 721.174.T2 (T2). Peptides eluted from allogeneic HLA-A2-matched RCC and melanoma cell lines exhibited the CTL-5-defined epitope in the same HPLC fractions as peptides derived from the autologous RCC line. Renal cancer and melanoma cells preincubated with interferon-gamma (IFN-gamma) resulted in an additional peak of reconstitution activity in both cell types. This second lytic peak was also observed when high amounts of autologous RCC cells were used for peptide preparation without IFN-gamma pretreatment, indicating that IFN-gamma increases the amount of MHC class I/peptide complexes per cell, rather than inducing a neo-epitope.

B-Lymphocytes↗
PubMed1996

Multiple levels of MHC class I down-regulation by ras oncogenes.

A number of tumours and oncogene transformed cells displayed reduced MHC class I surface expression which seemed to enable their escape from immune surveillance. To test whether oncogenic activation is directly involved in suppressing MHC class I expression, a model of inducible oncogene expression was chosen. Mouse fibroblasts transfected with different oncogenes expressed under the control of the dexamethasone-inducible MMTV promoter were analysed in the presence and absence of hormone for the mRNA and protein expression of MHC class I molecules as well as the respective oncogenes. Immunofluorescence analyses demonstrated an inverse association of MHC class I and oncogene expression after dexamethasone stimulation, independent of the type of oncogene causing transformation. Hormone-mediated induction of oncogene expression caused down-regulation of all H-2 loci. Kinetic experiments using MMTV c-Ha-ras(A) transfectants revealed that down-regulation of MHC class I surface expression was preceded by a dexamethasone-induced change of morphology, anchorage-independent growth, and an increase of the ras protein p 21. Parallel monitoring of mRNA expression demonstrated a time-dependent up-regulation of ras specific transcripts, which was associated with differential regulation of MHC class I heavy and light chain transcripts. Beta2-microglobulin transcripts were transiently suppressed, whereas MHC class I heavy chain transcripts remained unaffected. To investigate the mechanisms of oncogene-mediated down-regulation of MHC class I expression, H-2 promoter transfections and a nuclear run on assays were performed. In MMTV c-Ha-ras(A) cells, neither alterations of the H-2 promoter activity nor of the transcriptional activity of H-2 antigens was observed in the presence of dexamethasone, whereas both could be up-regulated by interferon-gamma treatment. These data suggest that oncogene-mediated transformation is directly associated with MHC class I down-regulation, but that complex interactions affecting MHC class I heavy and light chain genes at the transcriptional and/or post-transcriptional level are involved in this process.

3T3 Cells↗
PubMed1996

[High dose chemotherapy in treatment of breast carcinoma: current status and limits of this method of therapy].

Current Status and Limits: High-dose chemotherapy with autologous haematopoietic stem cell rescue has become in recent years a widely accepted therapeutic modality for advanced stage breast cancer in North America. The emergence of modern supportive measures like peripheral blood stem cell rescue has significantly decreased the toxicity and cost of high-dose chemotherapy. The rationale for use of escalated chemotherapy doses in breast cancer is the establishment of a dose-response relationship, with higher doses producing increased response rates in preclinical studies as well as in clinical trials. The dose limiting myelotoxicity of several active agents in breast cancer can only be overcome by rescue with previously cryopreserved autologous haematopoietic stem cells. Two phase II clinical trials with high-dose chemotherapy as adjuvant measure in high-risk breast cancer patients with multiple positive axillary nodes have been published so far. Disease-free survival is these 2 studies was 71 and 84% after 5 and 3 years of follow-up, and hence significantly longer compared to historic controls. Randomised phase III trials are urgently needed to confirm these promising results. Several reports of high-dose chemotherapy in disseminated breast cancer have been published so far. Most of these studies could establish a high response rate of about 70%, half of which were complete responses. However, most tumour regressions were short lived and overall survival was not prolonged compared with historic controls. Therefore, high-dose chemotherapy cannot be recommended for disseminated breast cancer patients outside of innovative clinical trials. To improve on the results in disseminated breast cancer patients the use of repeated cycles of high dose chemotherapy or the value of tumour cell purging of the stem cell product are being currently explored by different groups. In general, breast cancer patients should only be treated with high dose chemotherapy in context with a clinical trial.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1996

Immunomodulatory and hematopoietic effects of recombinant human interleukin-6 in patients with advanced renal cell cancer.

Interleukin-6 (IL-6) is a cytokine with pleiotropic biologic activities on B cells, T cells, and hematopoietic progenitors. The present study was undertaken to assess pharmacodynamic effects of subcutaneous administration of IL-6 on blood counts, immunologic parameters, and acute-phase reactants. Blood samples were taken from patients with advanced renal cell cancer participating in a phase II trial of recombinant human IL-6. Multiparameter FACS analyses of peripheral blood mononuclear cells were performed using antibodies against CD3, CD4, CD8, HLA-DR, CD56, CD28, CD38, CD19, sIgM, and sIgG. Serum levels of IL-10, soluble CD23 (sCD23), sCD25, IL-1 receptor antagonist protein (IL-1RA), soluble tumor necrosis factor (TNF) receptors (sTNF-R) p55 and p75, and soluble IL-6 receptor (sIL-6R) were detected by ELISA systems. Levels of C-reactive protein (CRP), neopterin, fibrinogen, beta 2-microglobulin, and immunoglobulins M, G, and A were measured by standard methods. In response to administration of IL-6, a significant increment in platelet counts was observed, reaching peak levels after 21 days of treatment. In contrast, leukocyte subsets remained unaffected. No change in number of immunophenotype of peripheral blood B cells, T cells, or natural killer cells could be detected following IL-6 administration. Blood levels of sCD23, IL-10, sIL-6R, neopterin, beta 2-microglobulin, and immunoglobulin subsets were not influenced by cytokine therapy. However, administration of IL-6 led to a slow increment of acute-phase reactants CRP and fibrinogen. Furthermore, the anti-inflammatory molecules sTNF-R p55 and p75 were induced by IL-6, whereas serum levels of IL-1RA remained unchanged. Finally, an increase in blood levels of sCD25 was observed. In conclusion, IL-6 in vivo predominantly acts as a regulator of inflammation and a megakaryocyte differentiation factor.

Acute-Phase Reaction↗
PubMed1996

Analysis of the p53 and MDM-2 gene in acute myeloid leukemia.

The MDM-2 (murine double minute 2) gene codes for a cellular protein that can bind to the p53 tumor suppressor gene product, thereby functioning as a negative regulator of p53. In order to define the role of the MDM-2 gene in the pathogenesis of human acute myeloid leukemia, the expression and the sequence of the MDM-2 gene were examined in samples of bone marrow and/or peripheral mononuclear cells of 38 patients by using immunostaining, polymerase chain reaction (PCR), single strand conformation polymorphism, and sequencing. Immunohistochemical staining detected a weak accumulation of the MDM-2 protein in AML patients of FAB classification M4 and M5. RT-PCR analysis revealed a heterogeneous expression pattern of MDM-2 mRNA in AML samples of different FAB classification. An increased level of MDM-2 mRNA expression was observed in 17 of 38 AML patients when compared to normal controls. No structural changes in a 488 bp region extending from nucleotide 890 to 1378 of the MDM-2 cDNA were detected using RT-SSCP and sequence analysis. In addition, heterogeneous expression of p53 transcripts was found with the highest p53 mRNA levels in AML M4 and M5. Interestingly, there seems to be a correlation between the relative ratios of p53 and MDM-2 mRNA levels in AML M4 and M5: in 15 of 23 cases high p53 mRNA expression was directly associated with high levels of MDM-2 transcripts. An exclusively intranuclear p53 immunostaining pattern was found in 10 of 16 (58%) AML FAB M4 and M5, whereas the remaining AML samples tested were negative for p53 (0/10). Using RT-SSCP analysis and direct sequencing of the RT-PCR amplification products of p53 exon 5-8, we observed that only 1 of 38 AML patients showed a point mutation in the p53 gene. This missense mutation occurred in the evolutionary highly conserved region of p53 at codon 255 (Ile to Phe). These data indicated that structural alterations of the p53 gene do not play an important role in the initiation and progression of AML. However, abrogation of p53 tumor suppressor function due to MDM-2 overexpression may be an alternative molecular mechanism by which a subset of AMLs may escape from p53-regulated growth control.

Acute Disease↗
PubMed1996

Influence of interferon-alpha on cytokine expression by the bone marrow microenvironment--impact on treatment of myeloproliferative disorders.

Myeloproliferative disorders (MPD) are characterized by several common clinical and biological features, although at the molecular level, each disease entity exhibits distinct abnormalities. IFN-alpha exerts beneficial therapeutic effects in chronic myelogenous leukemia, polycythemia vera and essential thrombocythemia, resulting in control of hematopoietic hyperplasia and, in a minority of patients, in induction of cytogenetic remission. The mechanism of action of IFN-alpha in MPD is poorly defined. Recently published in vitro findings suggest that IFN-alpha interacts with the regulation of hematopoiesis by multiple ways. Its antiproliferative activity is well known for more than a decade, however, substantial growth inhibition is achieved only at relatively high concentrations. Defective adhesion of hematopoietic progenitor cells in CML to bone marrow stromal cells is corrected by IFN-alpha, which might expose CML progenitors to inhibitory cytokines produced by the bone marrow microenvironment. Recent work from our group demonstrated, that IFN-alpha potently interacts with the production of hematopoietic cytokines in bone marrow stromal cells. Expression of stimulatory cytokines, such as GM-CSF, G-CSF, IL-1 and IL-11 is inhibited by IFN-ct, whereas the production of negative regulators, such as IL-1RA and MIP-1 alpha, is stimulated. The combined action of IFN-alpha on paracrine expression of cytokines suggests an indirect antihematopoietic effect, which might contribute to its clinical activity in MPD.

Adipose Tissue↗
PubMed1996

Inhibition of interleukin-11 by interferon-alpha in human bone marrow stromal cells.

Interleukin-11 (IL-11), which has been detected as a stromal cell-derived cytokine, regulates multiple steps of early hematopoiesis. Synthesis of IL-11 is induced by IL-1 and transforming growth factor-beta (TGF-beta) in stromal cells and fibroblasts, but the cytokines that inhibit its production remain to be defined. In the present study, we demonstrate that interferon-alpha (INF-alpha) downregulates IL-1-induced IL-11 in human bone marrow stromal cultures. In Northern blot experiments, expression of IL-11 mRNA was reduced in the presence of INF-alpha; this inhibiton was prevented by the addition of cycloheximide. In eight independent experiments, IL-1-stimulated production of IL-11 was significantly inhibited by INF-alpha (p = 0.0117) as measured in stromal cell supernatants by specific enzyme-linked immunosorbent assay (ELISA). Dose titration experiments revealed a dose-dependent inhibition already beginning at a dose level of 10 U/mL IFN. These results are in keeping with our previous observation defining an inhibitory role of INF-alpha in the production of hematopoietic growth factors produced by the bone marrow microenvironment.

Bone Marrow↗
PubMed1996

[Psychosomatic determinants of restenosis after percutaneous transluminal coronary angioplasty].

Coronary Angioplasty (PTCA) has become one of the standard procedures in the therapy of coronary heart disease. One of the main issues is the 20-40% incidence of restenosis of the dilated vessel. Up to now, the subject of predictors of restenosis has not yet been sufficiently clarified. Our study is concerned with two questions; first, is it possible to prove any connections between the amount of restenosis and psychosocial factors? Second, is it possible to distinguish groups of patients with and without subsequent restenosis on the basis of psychosocial variables, even before the diagnostic coronary angiography? 138 (91m, 47f) patients were assessed before the diagnostic coronarangiography, the 25 PTCA-patients (20m, 5f) were furthermore reassessed three months after the PTCA (the time of the angiographic control). Somatic and social data were collected and a semistandardised interview was conducted. In addition, coping, stress-coping control over disease and health and life-contentment were determined. Highly significant correlations was found between the amount of restenosis and resignative stress-coping, self-pity, depressive coping and flight-tendency. With the help of stress-coping-subtests (which were given at the first examination) it was possible to separate the group of patients with restenosis from the group without it.

Adaptation, Psychological↗
PubMed1996

Preliminary results of stem cell mobilization in chronic myeloid leukemia with a moderate intensity chemotherapy regimen and G-CSF or G-CSF plus IL-3.

Mobilization of Philadelphia chromosome (Ph) negative peripheral blood stem cells has been reported subsequent to intensive chemotherapy. We asked whether peripheral blood stem cells can be harvested subsequent to a less toxic chemotherapy regimen. Patients were treated with idarubicin 12 mg/m2 on day 1 and 2 and ara-C 100 mg/m2 days 1-5 and 5 or 10 micrograms/m2 G-CSF. In case of insufficient yield chemotherapy was repeated using IL-3 and G-CSF for mobilization of stem cells. Fourteen patients received 18 cycles of chemotherapy. The majority of patients were in late chronic phase and treated after secondary (interferon-alpha) IFN-alpha resistance. sufficient numbers of peripheral blood stem cells were harvested in 11 out of 14 patients. Although mixed Ph positive/Ph negative leukaphereses were harvested in the majority of patients, in no case were sufficient numbers of purely Ph negative progenitor cells for transplantation obtained. No toxic deaths were observed during the aplasia and the toxicity was acceptable. These preliminary results demonstrate that this procedure can be safely applied in patients with chronic phase CML and allows the harvesting of sufficient numbers of peripheral blood stem cells. The efficacy of this regimen for the mobilization of Ph negative cells should be further explored in patients at an earlier stage of the disease.

Adult↗
PubMed1996

Overexpression of mouse p140 subunit of replication factor C accelerates cellular proliferation.

Screening of a murine cDNA expression library with an IFN-stimulated response element (ISRE), as a recognition site DNA probe, resulted in the isolation of a cDNA encoding a polypeptide of 1145 amino acids designated ISRE-binding factor-1. This was subsequently shown to be identical to the M(r) 140,000 subunit of replication factor C (RFC). RFC is required, along with the proliferating cell nuclear antigen and DNA polymerase delta, for the synthesis of the leading strand during DNA replication. RFC exhibits a structure-specific DNA-binding activity that has been localized to its M(r) 140,000 subunit (p140). Sequence-specific binding of this polypeptide to the ISRE occurs only with low affinity. Based on DNA-binding activity of the truncated RFC-p140 encoded by the partial cDNA isolated, the DNA-binding domain of this polypeptide was mapped to a region encoded by amino acids 366 to 540. Transfection of NIH 3T3 cells with an expression plasmid containing murine RFC-p140 driven by cytomegalovirus early promoter led to the establishment of stable cell lines that expressed a 2.5- to 3.0-fold higher RFC-p140 protein level in comparison with control cells. The stable clones exhibited significantly accelerated cell proliferation, indicating that RFC-p140 is the limiting subunit of an active RFC complex in normal cells.

3T3 Cells↗
PubMed1996

Suppression of MHC class I antigens in oncogenic transformants: association with decreased recognition by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) recognize peptide fragments derived from endogenous proteins, processed internally, and presented at the cell surface by major histocompatibility complex (MHC) class I molecules. The use of specific CTL for cancer therapy is limited because of their dependence on effective processing and presentation of appropriate antigenic peptides. Structural alterations, like point mutation or somatic loss, or dysregulation of key elements in the processing or presentation pathway, may allow cells to escape the immune surveillance. Indeed, the expression of MHC class I antigens on the surface of virus- and oncogene-transformed cells is low and correlates with tumorigenicity. Transformation of murine fibroblasts with the ras oncogene results in the suppression of cell surface expression of all H-2 loci as determined by FACScan analysis using a panel of monoclonal antibodies. We then examined whether the oncogene-mediated suppression of MHC class I surface expression was associated with reduced recognition of transformants by CD8+ T lymphocytes. Murine T lymphoma cells were stably transfected by the Ha-ras oncogene. The transfectants expressed distinct levels of the Ha-ras specific protein p21. Again, immunofluorescence analysis demonstrated an inverse correlation between oncogene and MHC class I surface expression in RMAras transformants. Allogeneic H-2Kb-restricted cytotoxic T lymphocytes were able to efficiently lyse the parental T lymphoma cells. In contrast, the CTL-mediated lysis of ras transformants was significantly downregulated compared with untransfected RMA cells. The efficiency of CTL-mediated lysis of RMAras cells was directly associated with reduced MHC class I membrane and high p21ras protein expression. Thus, the oncogene-mediated downregulation of MHC class I surface expression resulted in a reduced CTL response. Attempts are in progress to revert the defects in MHC class I surface expression of oncogenic transformants by introducing the different elements of the antigen presentation pathway. Such studies will not only provide improved understanding of the mechanisms of tumor escape, but also will suggest strategies to repair cellular defects in cancer patients having impaired expression of MHC class I antigens.

3T3 Cells↗