Éva Oláh

Effect of frequently used chemotherapeutic drugs on the cytotoxic activity of human natural killer cells

Tumors are considered to be possible targets of immunotherapy using stimulated and expanded autologous or allogeneic natural killer (NK) cells mismatched for MHC class I molecules and inhibitory NK receptors. NK cell–based immunoadjuvant therapies are carried out in combination with standard chemotherapeutic protocols. In the presented study, we characterized the effect of 28 frequently used chemotherapeutic agents on the capacity of NK cells to kill target cells. We found that treatment of NK cells with the drugs vinblastine, paclitaxel, docetaxel, cladribine, chlorambucil, bortezomib, and MG-132 effectively inhibited NK cell–mediated killing without affecting the viability of NK cells. On the other hand, the following drugs permitted efficient NK cell–mediated killing even at concentrations comparable with or higher than the maximally achieved therapeutic concentration in vivo in humans: asparaginase, bevacizumab, bleomycin, doxorubicin, epirubicin, etoposide, 5-fluorouracil, hydroxyurea, streptozocin, and 6-mercaptopurine. [Mol Cancer Ther 2007;6(2):644–54]

High-throughput live-cell imaging reveals differential inhibition of tumor cell proliferation by human fibroblasts

Increasing evidence indicates that cancer development requires changes both in the precancerous cells and in their microenvironment. To study one aspect of the microenvironmental control, we departed from Michael Stokers observation (Stroker et al, J Cell Sci 1966;1:297–310) that normal fibroblasts can inhibit the growth of admixed cancer cells (neighbour suppression). We have developed a high‐throughput microscopy and image analysis system permitting the examination of live mixed cell cultures growing on 384‐well plates, at the single cell level and over time. We have tested the effect of 107 samples of low passage number (<5) primary human fibroblasts from pediatric and adult donors, on the growth of six human tumor cell lines. Three of the lines were derived from prostate carcinomas, two from lung carcinomas and one was an EBV‐transformed lymphoblastoid line. Labeled tumor cells were grown in the presence of unlabeled fibroblasts. The majority of the tested fibroblasts inhibited the proliferation of the tumor cells, compared to the control cultures where labeled tumor cells were co‐cultured with unlabeled tumor cells. The proliferation inhibiting effect of the fibroblasts differed depending on their site of origin and the age of the donor. Inhibition required direct cell contact. Mouse 3T3 fibroblasts inhibited the growth of SV40‐transformed 3T3 cells and human tumor cells, showing that the inhibitory effect could prevail across the species barrier. Our high‐throughput system allows the quantitative analysis of the inhibitory effect of fibroblasts on the population level and the exploration of differences depending on the source of the normal cells.

Abnormalities of chromosome 1 in relation to human malignant diseases

Chromosome 1 is known to often be involved in various malignant diseases. Its numerical and structural aberrations have been observed in chronic and acute leukemias and solid tumors as well. Recently five protooncogenes have been assigned to the long and short arms of chromosome 1. The frequent and nonspecific occurrence of chromosome 1 rearrangements in human tumors suggests that they play an important role in the pathogenesis and progression of these diseases. The frequency, types, and time of the occurrence of chromosome 1 aberrations and their relation to the stage of the disease were studied in 317 patients with various malignant diseases. In ten patients nonrandom aberrations of chromosome 1 were observed. Two patients had CML, two PRV followed by ANLL, and the remaining six patients suffered from ANLL, ALL, Burkitt lymphoma, MF, SMMoL, and IRSA, respectively. In six patients, total or partial trisomy of the long arm or of the whole chromosome 1 was present, and in three cases balanced translocations involving chromosome 1 could be found. In the cells of one patient a duplication of the centromeric heterochromatin was seen. We analyzed the breakpoints involved. Finally, the aberrations of chromosome 1 were almost always be observed at the terminal stage of the diseases.

Cystatin C is a suitable marker of glomerular function in children with cancer

Antineoplastic chemotherapy is associated with nephrotoxic side effects. Data on nephrotoxicity in childhood cancer are scanty, in part because of the difficulties in obtaining reliable markers of glomerular function. We used serum cystatin C (cysC) to assess glomerular function. CysC was compared with serum creatinine concentration (SCr), the endogenous creatinine clearence ( C Cr), and the calculated Counahan formula ( C Counahan) in children with leukemia and solid tumors. CysC was measured by particle-enhanced immunoturbidimetric assay. Serum and urinary creatinine concentrations were determined by the Jaffé method. Samples were obtained from 258 children, including 92 receiving anticancer chemotherapy, 108 long-term survivors, 40 children without any renal disease, and 18 patients with chronic renal insufficiency. CysC of patients on current chemotherapy was assessed both before and after treatment. Significant correlations were found between cysC and SCr and between 1/cysC and C Counahan. CysC increased significantly after cisplatin, methotrexate, cyclophosphamide, ifosfamide, and multimodality treatment. Our results suggest that cysC measurement can be used to characterize glomerular function in children with cancer.

Alterations of P53 and RB genes and the evolution of the accelerated phase of chronic myeloid leukemia.

Using the single-strand conformation polymorphism and heteroduplex analyses, the P53 and RB genes were analyzed in cell samples from twenty-eight patients with chronic myeloid leukemia (CML) both at diagnosis and at the onset of accelerated phase (AP) of the disease. No alterations of the P53 or RB genes were found in any of the chronic phase (CP) samples. Structural abnormalities of the P53 gene were observed in ten of twenty-eight AP samples within exons 4, 5, 7 and 9. Of the ten cases of AP disease with altered P53 genes, five patients also suffered from the deletion of the other allele. Alterations of the RB gene could be detected in six AP samples, and aberrant band patterns were found in the analysis of exons 2, 3, 4, 6, 7, 13, 14, 17, 21 and 26. Among the six AP samples with structural abnormalities of the RB gene, two showed the loss of the other allele. It is of note that alterations of both P53 and RB genes were observed in two AP samples. Our data strongly suggest that abnormalities of the P53 and RB genes and acceleration of CML are linked events in some cases of AP.

Phenotypic manifestations of the OCTN2 V295X mutation: sudden infant death and carnitine-responsive cardiomyopathy in Roma families.

In two non‐consanguineous Hungarian Roma (Gypsy) children who presented with cardiomyopathy and decreased plasma carnitine levels, we identified homozygous deletion of 17081C of the SLC22A5 gene that results in a frameshift at R282D and leads ultimately to a premature stop codon (V295X) in the OCTN2 carnitine transporter. Carnitine treatment resulted in dramatic improvement of the cardiac symptoms, echocardiographic, and EKG findings in both cases. Family investigations revealed four sudden deaths, two of them corresponded to the classic SIDS phenotype. In postmortem tissue specimens available from three of them we could verify the homozygous mutation. In liver tissue reserved from two patients lipid droplet vacuolization could be observed; the lipid vacuoles were located mainly in the peripherolobular regions of the acini. In the heart tissue signs of generalized hypertrophy and lipid vacuoles were seen predominantly in the subendocardial areas in both cases; some aggregates of smaller lipid vacuoles were separated, apparently by membranes. Review of all OCTN2 deficiency cases reported so far revealed that this is the first presentation of histopathology in classic familial sudden infant death syndrome (SIDS) with an established SLC22A5 mutation. In addition to the two affected homozygous cardiomyopathic children and three homozygous sudden death patients, the genetic analysis in 25 relatives showed 14 carriers. The mutant gene derived from five non‐consanguineous grandparents, each of them having 6–14 brothers and sisters. This alone suggests a wide ancestral spread of the mutation in certain Roma subpopulations.

Diagnostic significance of HLA-DQ typing in patients with previous coeliac disease diagnosis based on histology alone

Coeliac disease is strongly associated with human leukocyte antigen (HLA)‐DQ2 or DQ8 genotypes. The diagnosis is based on demonstrating crypt‐hyperplastic villous atrophy, endomysial or transglutaminase antibodies and correlation of disease activity with gluten intake.

Effect of frequently used chemotherapeutic drugs on cytotoxic activity of human cytotoxic T-lymphocytes.

Tumors are considered to be possible targets of immunotherapy using stimulated and expanded cytotoxic T-lymphocytes (CTL). It is important to consider the drug-induced effects when chemotherapeutic regimens and CTL-mediated immunotherapy is planned to be used in parallel. In this study, we characterized the effect of 29 frequently used chemotherapeutic agents on the cytotoxic activity of autologous and allogeneic CTLs. We found that treatment of CTLs with the following drugs: docetaxel, vincristine, chlorambucil, mitomycin C, oxaliplatin, doxorubicin, and bleomycin effectively inhibited CTL-mediated killing, without affecting their viability. On the other hand, the following drugs enhanced or permitted efficient CTL-mediated killing in vitro at concentrations comparable with the maximally achieved therapeutic concentration in vivo in humans: daunorubicin, prednisolone, vinorelbine, cisplatin, methotrexate, hydroxyurea, cytarabine, cyclophosphamide, topotecan, epirubicin, fluorouracil, carboplatin, asparaginase, 6-mercaptopurine, and bortezomib. Our results could potentially be used in the future to design new CTL-based adjuvant immunotherapy protocols.

Frequent methylation of p16INK4A and p14ARF genes implicated in the evolution of chronic myeloid leukaemia from its chronic to accelerated phase.

The frequency and mechanism of p16(INK4A) and p14(ARF) gene alterations were studied in cell samples from 30 patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukaemia (CML), both at diagnosis and at the onset of the accelerated phase (AP) of the disease. No alterations in the p16(INK4A) or p14(ARF) genes were found in any of the chronic phase (CP) samples. DNA sequencing analyses detected p16(INK4A) or p14(ARF) mutations in 17 AP samples. All mutations were heterozygous without loss of the other allele. Aberrant methylation of the p16(INK4A) or p14(ARF) promoters was found in 14 of 30 AP samples. The most common situation was the simultaneous methylation of both promoters. Our data indicate that p16(INK4A) and p14(ARF) are primary targets for inactivation by promoter methylation in the acceleration of CML. Transcriptional silencing of the p16(INK4A) and p14(ARF) genes may be important in the conversion of CML from the CP to the AP.

Myosin phosphatase interacts with and dephosphorylates the retinoblastoma protein in THP-1 leukemic cells: Its inhibition is involved in the attenuation of daunorubicin-induced cell death by calyculin-A

Reversible phosphorylation of the retinoblastoma protein (pRb) is an important regulatory mechanism in cell cycle progression. The role of protein phosphatases is less understood in this process, especially concerning the regulatory/targeting subunits involved. It is shown that pretreatment of THP-1 leukemic cells with calyculin-A (CL-A), a cell-permeable phosphatase inhibitor, attenuated daunorubicin (DNR)-induced cell death and resulted in increased pRb phosphorylation and protection against proteolytic degradation. Protein phosphatase-1 catalytic subunits (PP1c) dephosphorylated the phosphorylated C-terminal fragment of pRb (pRb-C) slightly, whereas when PP1c was complexed to myosin phosphatase target subunit-1 (MYPT1) in myosin phosphatase (MP) holoenzyme dephosphorylation was stimulated. The pRb-C phosphatase activity of MP was partially inhibited by anti-MYPT1(1-296) implicating MYPT1 in targeting PP1c to pRb. MYPT1 became phosphorylated on both inhibitory sites (Thr695 and Thr850) upon CL-A treatment of THP-1 cells resulting in the inhibition of MP activity. MYPT1 and pRb coprecipitated from cell lysates by immunoprecipitation with either anti-MYPT1 or anti-pRb antibodies implying that pRb-MYPT1 interaction occurred at cellular levels. Surface plasmon resonance-based experiments confirmed binding of pRb-C to both PP1c and MYPT1. In control and DNR-treated cells, MYPT1 and pRb were predominantly localized in the nucleus exhibiting partial colocalization as revealed by immunofluorescence using confocal microscopy. Upon CL-A treatment, nucleo-cytoplasmic shuttling of both MYPT1 and pRb, but not PP1c, was observed. The above data imply that MP, with the targeting role of MYPT1, may regulate the phosphorylation level of pRb, thereby it may be involved in the control of cell cycle progression and in the mediation of chemoresistance of leukemic cells.