p53 is associated with high-risk and pinpointsTP53missense mutations in mantle cell lymphoma

Survival for patients diagnosed with mantle cell lymphoma (MCL) has improved drastically in recent years. However, patients carrying mutations in tumour protein p53 (TP53) do not benefit from modern chemotherapy-based treatments and have poor prognosis. Thus, there is a clinical need to identify missense mutations through routine analysis to enable patient stratification. Sequencing is not widely implemented in clinical practice for MCL, and immunohistochemistry (IHC) is a feasible alternative to identify high-risk patients. The aim of the present study was to investigate the accuracy of p53 as a tool to identify patients withTP53missense mutations and the prognostic impact of overexpression and mutations in a Swedish population-based cohort. In total, 317 cases were investigated using IHC and 255 cases were sequenced, enabling analysis of p53 andTP53status among 137 cases divided over the two-cohort investigated. The accuracy of predicting missense mutations from protein expression was 82%, with sensitivity at 82% and specificity at 100% in paired samples. We further show the impact of p53 expression andTP53mutations on survival (hazard ratio of 3 center dot 1 in univariate analysis for both), and the association to risk factors, such as high MCL International Prognostic Index, blastoid morphology and proliferation, in a population-based setting.Peer reviewe

Health Promoting Effects of Bioactive Peptides in Milk - Studies on Cultured Normal Cells and Cancer Cells

This thesis concerns the bioactive peptides lactoferricin and ubiquitin and findings suggesting that they have ability to modify the risk of cancer. It is known that components in bovine milk may improve human health and modify the risk of diseases. Examples of such compounds are bioactive peptides, i.e. peptides with an effect on the body beyond the purely nutritional. Using cell culture as a model system possible cancer preventing effects were investigated with focus on cell proliferation, cell death and DNA repair. In one study, the effect of lactoferricin on CaCo-2 human colon cancer cells was investigated. Low concentrations of the peptide, equivalent to the amount released in the gut of a milk consumer, were used to study the effects during several weeks. A decreased proliferation rate was observed in cancer cells, raising the hypothesis that a prolongation of the cell cycle may be beneficial for the DNA repair system in normal cells. To test this, the effect of lactoferricin treatment was studied in a normal colon cell line. Lactoferricin treatment resulted in decreased proliferation and decreased levels of G1 and S phase proteins. In another study we induced DNA damage in CaCo-2 cells by exposure to UV-light and allowing them to grow in the absence or presence of lactoferricin. The measurement of DNA damage, as detected by the alkaline Comet assay, showed that cells treated with lactoferricin had less DNA damage than the control cells. Another part of the work was to identify new bioactive peptides in milk. Using quantitative Western blot and peptide mass fingerprinting techniques we found that the peptide ubiquitin is present in milk in low concentrations. The effect of ubiquitin on cell proliferation and protein expression was examined both in CaCo-2 cells and in two neuroblastoma cell lines, SH-SY5Y and LA-N-1. Ubiquitin treatment decreased the cell proliferation rate in CaCo-2 and SH-SY5Y cells. LA-N-1 cells initially responded to the ubiquitin treatment with a decreased proliferation rate but after almost three weeks of treatment an alteration in the growth pattern took place and the treated cells grew faster than the control cells. The cell proliferative effects correlated with the level of ubiquitination of intracellular proteins observed in respective cell line. The results showed that the effects of lactoferricin and ubiquitin on cell proliferation may contribute to the health promoting effects of milk

Reduction of ultraviolet light-induced DNA damage in human colon cancer cells treated with a lactoferrin-derived peptide

Treatment of Caco-2 cells with the peptide lactoferricin(4-14), results in reduction of the growth rate by prolongation of the S phase of the cell cycle. Lactoferricin(1-25) is formed in the gut by cleavage from lactoferrin and the bioactive amino acids are found within lactoferricin(4-14). Our hypothesis is that the reduction of the rate of S phase progression may result in increased DNA repair. To test this hypothesis, Caco-2 cells were subjected to UV light that caused DNA lesions and then the cells were grown in the absence or presence of 2.0 mu M lactoferricin(4-14). Evaluation of DNA strand breaks using the comet assay showed that lactoferricin(4-14) treatment indeed resulted in a reduction of comets showing damaged DNA. In the search for a mechanism, we have investigated the levels of several proteins involved in cell cycle regulation, DNA replication, and apoptosis using Western blot. Lactoferricin(4-14) treatment resulted in an increased expression of flap endonuclease-1 pointing to increased DNA synthesis activity. Lactoferricin(4-14) treatment decreased the expression of the proapoptotic protein B-cell lymphoma 2-associated X protein (or Bax), indicating decreased cell death. As we have found previously, lactoferricin(4-14) treatment reduced the expression of cyclin E involved in the G(1)/S transition. Immunofluorescence microscopy showed that a lower gamma-H2AX expression in lactoferricin(4-14)-treated cells, pointing to more efficient DNA repair. Thus, altogether our data show that lactoferricin(4-14) treatment has beneficial effects

Ibrutinib inhibits antibody dependent cellular cytotoxicity induced by rituximab or obinutuzumab in MCL cell lines, not overcome by addition of lenalidomide

Background: The Bruton's Tyrosine Kinase (BTK)-inhibitor ibrutinib is highly active in mantle cell lymphoma (MCL) but may inhibit response to anti-CD20 antibody as previously shown in CLL models. We investigated how antibody-dependent cellular cytotoxicity (ADCC) induced by type I/II anti-CD20 antibodies was affected by treatment with ibrutinib in MCL. Furthermore, we investigated if lenalidomide, a potential sensitizer to anti-CD20 treatment, could prevent an inhibitory effect of ibrutinib. Methods: Anti-CD20 (rituximab/obinutuzumab) opsonized MCL cell lines were co-cultured with ibrutinib (± lenalidomide) - exposed effector cells, and analyzed for evaluation of cell death. Results: Cell death induced by rituximab was reduced with 75% at 0.5 μM ibrutinib and with 52% at 0.1 μM ibrutinib when induced by obinutuzumab, even by addition of lenalidomide. Moreover, obinutuzumab was associated with higher rate of cell death compared to rituximab. Conclusion: Ibrutinib negatively affects anti-CD20 induced cell death in MCL, not reversed by lenalidomide. Explorations of sequential administration and selective BTK-inhibitors may reveal the optimal combination of novel agents in MCL

Lactoferricin treatment decreases the rate of cell proliferation of a human colon cancer cell line.

Food components modify the risk of cancer at a large number of sites but the mechanism of action is unknown. In the present investigation, we studied the effect of the peptide lactoferricin derived from bovine milk lactoferrin on human colon cancer CaCo-2 cells. The cells were either untreated or treated with 2.0, 0.2, or 0.02 microM lactoferricin. Cell cycle kinetics were investigated with a bromodeoxyuridine DNA flow cytometric method. The results show that lactoferricin treatment slightly but significantly prolonged the S phase of the cell cycle. Lactoferricin treatment lowered the level of cyclin E1, a protein involved in the regulation of genes required for G(1)/S transition and consequently for efficient S phase progression. The slight prolongation of the S phase resulted in a reduction of cell proliferation, which became more apparent after a long treatment time

Identification of ubiquitin in bovine milk and its growth inhibitory effects on human cancer cell lines.

Bovine milk is associated with improved health and reduced risk of several diseases, among them cancer. Milk is a complex mixture of known and unknown components. The components and the mechanisms that contribute to the cancer-preventive effects are largely unknown. We set out to find new peptides in milk and identified ubiquitin (Ub) using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Western blot. Using quantitative Western blot, we estimated the Ub concentration to be about 0.003 micromol/L in milk. We then decided to investigate the effect of treating human colon cancer CaCo-2 cells with Ub, using higher concentrations than in milk. CaCo-2 cells treated with 0.02 to 2.0 micromol/L Ub showed significantly decreased proliferation compared with untreated control cells. A higher growth inhibitory effect than in CaCo-2 cells was found in the neuroblastoma cell line SH-SY5Y treated with 0.02 to 0.2 micromol/L Ub. A bromodeoxyuridine DNA flow cytometric method was used to study cell cycle kinetics in Ub-treated CaCo-2 cells. The data point toward a prolongation of the G(1) phase. The levels of several cell cycle regulatory proteins were affected. Our data point to Ub possibly being one of the components in milk reducing the risk of cancer

Suppression of Secondary Antibody Response by Intravenous Immunoglobulin in a Patient with Haemophilia B and Antibodies

A 39‐year‐old patient, suffering from severe haemophilia B and antibodies against factor IX, has twice been treated with extracorporeal protein A‐Sepharose adsorption followed by conventional substitution therapy in combination with immunosuppression (cyclophos‐phamide). On both occasions, separated by a 2‐year interval, the same procedure was followed except that, on the second, administration of i.v. immunoglobulin (Gammonativ, KabiVitrum) was added. Within a week of the first treatment the patient developed a 15‐fold increase in the antibody titre. Following the second treatment described here, no secondary antibody response could be detected, and after a further 12 weeks only traces of antibodies are demonstrable. It seems that antibody synthesis was suppressed by the i.v. immunoglobulin. No evidence was found to demonstrate that the effect was due either to a non‐specific suppression of the immune and reticuloendothelial systems or to the action of interfering antibodies. It has not yet been established whether or not the protein A‐Sepharose adsorption technique, or the immunosuppressive treatment, contributed in any way to the result. The observations suggest a new approach to the treatment of haemophiliacs with antibodies of the high‐responding type

Bortezomib prevents cytarabine resistance in MCL, which is characterized by down-regulation of dCK and up-regulation of SPIB resulting in high NF-κB activity

BACKGROUND: The addition of high-dose cytarabine to the treatment of mantle cell lymphoma (MCL) has significantly prolonged survival of patients, but relapses are common and are normally associated with increased resistance. To elucidate the mechanisms responsible for cytarabine resistance, and to create a tool for drug discovery investigations, we established a unique and molecularly reproducible cytarabine resistant model from the Z138 MCL cell line.METHODS: Effects of different substances on cytarabine-sensitive and resistant cells were evaluated by assessment of cell proliferation using [methyl-14C]-thymidine incorporation and molecular changes were investigated by protein and gene expression analyses.RESULTS: Gene expression profiling revealed that major transcriptional changes occur during the initial phase of adaptation to cellular growth in cytarabine containing media, and only few key genes, including SPIB, are deregulated upon the later development of resistance. Resistance was shown to be mediated by down-regulation of the deoxycytidine kinase (dCK) protein, responsible for activation of nucleoside analogue prodrugs. This key event, emphasized by cross-resistance to other nucleoside analogues, did not only effect resistance but also levels of SPIB and NF-κB, as assessed through forced overexpression in resistant cells. Thus, for the first time we show that regulation of drug resistance through prevention of conversion of pro-drug into active drug are closely linked to increased proliferation and resistance to apoptosis in MCL. Using drug libraries, we identify several substances with growth reducing effect on cytarabine resistant cells. We further hypothesized that co-treatment with bortezomib could prevent resistance development. This was confirmed and show that the dCK levels are retained upon co-treatment, indicating a clinical use for bortezomib treatment in combination with cytarabine to avoid development of resistance. The possibility to predict cytarabine resistance in diagnostic samples was assessed, but analysis show that a majority of patients have moderate to high expression of dCK at diagnosis, corresponding well to the initial clinical response to cytarabine treatment.CONCLUSION: We show that cytarabine resistance potentially can be avoided or at least delayed through co-treatment with bortezomib, and that down-regulation of dCK and up-regulation of SPIB and NF-κB are the main molecular events driving cytarabine resistance development