The present study was performed to gain insight into the role of p53 and p21(WAF1) on the cytotoxicity of the purine analogue cladribine (2-CdA) on cancer cells. Drug sensitivity, cell cycle ...distribution and drug-induced cell death were compared in three lines derived from the colorectal carcinoma HCT116: the p53+/+ cell line containing wild-type p53 and the p53-/- and p21(WAF1)-/- lines, in which both alleles of p53 or p21(WAF1) were deleted by homologous recombination, respectively. p53-/- and p21(WAF1)-/- cells were significantly more resistant to the cytotoxic effects of 2-CdA than the p53+/+ cells. p53+/+ cells and p21(WAF1)-/-, but not p53-/- cells, displayed wt-p53 protein accumulation and arrested in S-phase after exposure to 2-CdA. mRNA analysis of the transporter hENT1 and of enzymes involved in drug metabolism did not show alterations which might explain a drug-resistant phenotype in the p53-/- or p21(WAF1)-/- cells. Exposure of p53+/+ cells to 2-CdA resulted in expression of p21(WAF1) mRNA and protein, enhanced expression of uncleaved PARP-1, and a higher degree both of apoptosis and necrosis than in p53-/- and p21(WAF1)-/- cells exposed to 2-CdA. Addition of the specific PARP-1 inhibitor 3-AB to 2-CdA-treated cells rendered p53+/+ cells resistant to this drug. Bax levels were reduced in the p53-/- while they increased in the p53+/+ line and remained stable in the p21(WAF1)-/- cells. We conclude that p53 and p21(WAF1) status of cancer cells influences their sensitivity to 2-CdA cytotoxicity. This may involve alterations in the apoptotic cascade as well as in PARP-1-dependent cell death.
Canertinib pfizer Galmarini, Carlos Maria
IDrugs : the investigational drugs journal
7, Številka:
1
Journal Article
Canertinib, a water-soluble, orally available analog of PD-169414 (Pfizer Inc), is an EGFR tyrosine kinase inhibitor under development by Pfizer Inc as a potential treatment for cancer.
We analyzed cytosolic 5'-(3')-nucleotidase (dNT-1) mRNA expression by quantitative polymerase chain reaction at diagnosis in leukemic blasts from 114 patients with acute myeloid leukemia (AML) ...treated with ara-C. Our results show that low dNT-1 mRNA expression in leukemic blasts at diagnosis is correlated with a worse clinical outcome and suggest that this enzyme may have a role in sensitivity to ara-C in AML patients.
Abstract 3792
Poster Board III-728
p53 mutation is the most frequent single genetic abnormality found in therapy related AML and is also quite frequent in de novo AML with an incidence between 10% ...and 25%; Moreover patients with p53 mutations characteristically present complex karyotypes and complicated chromosome rearrangements, leading to an adverse prognosis and resistance to conventional chemotherapeutic agents. Consequently, there is a need for novel antileukemic drugs that could work on both p53-wild type and p53-mutated AML patients. In this study we have evaluated the activity of Zalypsis, a novel alkaloid from marine origin, in several AML cell lines and patients samples with different p53 status.
The efficacy of Zalypsis was analyzed in four AML cell lines (HL60, HEL, MV4.11 and KG1) and in cells from fresh bone marrow samples from ten newly diagnosed AML patients. The cytotoxicity was analyzed by means of MTT assay and with a multiparametric flow cytometry (MFC) technique that allowed us to study the efficacy of the drug in both mature and immature blast cell compartments. Proteomic and genomic changes induced after treatment with Zalypsis were analyzed in two AML cell lines with different p53 status (HEL and HL60) by Western-Blot and gene expression profile studies.
Zalypsis showed a very potent antileukemic activity in the four AML cell lines tested, with IC50s at 48 hours below 1 nM. When compared with the in vitro activity of conventional antileukemic agents, such as cytarabine, doxorubicine or fludarabine, Zalypsis turned out to be 10-100 times more potent. It also showed remarkable ex vivo potency in freshly isolated blasts from ten AML patients. In these patients samples, we had the opportunity to separately analyze by multiparametric flow cytometry, the activity of Zalypsis in the different blasts populations, and we constantly observed similar activity in the most mature blast population and in the most immature one (CD34+, CD38- Lineage-), which is thought to include the leukemic stem cells and which is usually more resistant to conventional chemotherapy. Regarding toxicity, Zalypsis preserved the CD34+ non tumoral hematopoietic progenitor cells. The combination of this novel drug with conventional antileukemic agents indicated that Zalypsis is a good partner for combination with all of them, being the combination with cytarabine and daunorubicin particularly attractive for the clinic. Interestingly, as already said, all cell lines were extremely sensitive to Zalypsis, independently of the p53 status, as some of them displayed high basal levels of this protein by Western-Blot and had mutated p53 (HEL and MV 4.11), whereas its protein expression in the remaining cell lines was low, with KG1 bearing a mutation of the gene and HL60 a p53 deletion. Nevertheless, these last cells with low levels of the protein were a bit more sensitive to the drug, pointing out to a role of p53 in Zalypsis induced cell death. This was in agreement with a clear induction of DNA double strand breaks after treatment with Zalypsis, which was evidenced by an increase in phospho Histone H2AX, phospho CHK1 and phospho CHK2 after treatment of both HEL and HL60 with this compound. This was followed by the overexpression of p53 in the AML cell line bearing low basal levels of this protein (HL60). These results were further confirmed in the gene expression profile studies, in which treatment of AML cells with Zalypsis resulted in an important deregulation of genes involved in DNA damage response, including genes implicated in the ATM repair pathway and other mRNAs related to DNA repair, such as TLK2, ATR, ATMIN, CHEK2, RAD5, RAD52, RAD54L, BRCA1, BRCA2 and GADD45B among others. This response ended up in a clear induction of apoptosis as assessed by annexin V studies, caspase and PARP cleavage with partial rescue of cell death with the caspase inhibitor ZVAD-FMK, DNA laddering and loss of mitochondrial membrane potential analyzed by DiOC6 with the subsequent release of cytochrome C and AIF into the cytosol.
The potent and selective antileukemic effect observed with Zalypsis in AML cell lines and in patient's samples through a DNA damage response, together with its activity in both p53 mutated- and deleted-cells, provides the rationale for the investigation of Zalypsis in clinical trials for patients with AML.
Galmarini:Pharmamar: Employment. Avilés:Pharmamar: Employment. Cuevas:Pharmamar: Employment. Pandiella:Pharmamar: Research Funding. San-Miguel:Pharmamar: Research Funding.
Background and Aims: Although recent therapeutic advances have led to an improvement in the outcome of Multiple Myeloma (MM), it still remains an incurable disease, and therefore, new drugs with ...novel mechanisms of action are needed for myeloma patients. Zalypsis is a new synthetic alkaloid derived from certain marine compounds which has demonstrated significant in vitro and in vivo antitumor activity in different malignancies. It is currently under late Phase I development in solid tumours, with preliminary evidence of activity. In this study, we have analysed the preclinical activity and mechanism of action of Zalypsis in MM.
Material and methods: Nine different MM cell lines and BM samples from MM patients and normal donors were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and gene expression profile analysis. The in vivo activity was explored in a human subcutaneous plasmocytoma model and immunohistochemistry was performed in selected tumours.
Results: Zalypsis turned out to be the most potent antimyeloma agent we have tested so far in our laboratory, with IC50s in picomolar or low nanomolar ranges depending on the cell lines studied. Interestingly, the sensitivity to Zalypsis was independent of the pattern of resistance of the cell lines to conventional antimyeloma agents such as Dexamethasone or Melphalan. It also showed remarkable ex vivo potency in freshly isolated plasma cells from six patients (including two with plasma cell leukemia) and synergized with many other antimyeloma compounds, being the combination of Zalypsis + Lenalidomide + Dexamethasone particularly attractive. Regarding toxicity, Zalypsis preserved the CD34+ hematopoietic progenitor cells from MM and normal donor BM samples. This remarkable activity prompted us to investigate the mechanism of action of the drug. Besides the induction of apoptosis and cell cycle arrest, Zalypsis provoked DNA double strand breaks, which were evidenced by an increase in phospho Histone H2AX and phospho CHK2, followed by a striking overexpression of p53 in MM cell lines bearing wild type forms of this protein. Of note, no other compound currently used in the MM clinic induced such an increase in p53 protein levels. In addition, in a subset of MM cell lines in which p53 was mutated, Zalypsis also provoked DNA double strand breaks and induced cell death, although higher concentrations were required. Changes in the gene expression profile of MM cells treated with Zalypsis were concordant with these results, since important genes involved in DNA damage response were deregulated. This include genes implicated in the ATM repair pathway, such as TLK2, ATR, CHEK2, RAD5 and BRIP1 and other mRNAs related to DNA repair, such as RAD23B, XPC, XRCC1, XRCC5 and GADD45A. These results were confirmed in vivo in a model of human subcutaneous plasmocytoma in SCID mice. Zalypsis (0.8 and 1 mg/Kg) decreased tumour growth and improved survival of mice implanted with MM1S (wild type p53) and OPM-1 (mutated p53) plasmocytomas. Immunohistochemical studies in tumours from treated animals also demonstrated DNA damage with H2AX phosphorylation and p53 overexpression.
Conclusion: The potent in vitro and in vivo antimyeloma activity and the singular mechanism of action of Zalypsis uncovers the high sensitivity of tumour plasma cells to double strand breaks, and strongly supports the potential use of this compound in multiple myeloma patients.
Immunophenotyping has proven to be an interesting approach in patients with myelodysplastic syndromes (MDS)(Maynadie et al., Blood. 2002 Oct 1;100(7):2349–56). Most studies in these diseases have ...focused on the analysis of blast cells. We chose to determine the potential interest of markers expressed on other bone marrow compartments, namely granulocytes, monocytes and the CD34 compartment.
Fourteen marrow samples from patients with MDS and 10 normal bone marrow were analyzed. The median age in the MDS group and the control group was 64.4 and 59.8 years, respectively. Flow cytometry analysis of bone marrow samples was performed on a Facs Calibur, using four-color panels. The following antigens were analyzed: CD7, CD11c, CD14, CD15, CD16, CD33, CD34, CD45, CD36, CD38, CD56, CD71, CD117 and HLA-DR. The various compartments analyzed were defined using CD45/SSC gating, combined with SSC/CD34 gating for CD34+ cells. The median percentage of blasts in the MDS marrows was < 5%. Seven patients had a hemoglobin value < 100 g/dl. Five patients had a leukocyte count < 3.109/l. Six patients had a platelet count < 100.1012/L. Threshold values were determined by ROC analysis. Comparison of the expression levels of markers between MDS and normal bone marrows was performed using a Wilcoxon test. Analysis was performed on bone marrow granulocytes, monocytes and CD34 cells.
Granulocytes were found to express higher levels of CD33 in MDS than in normal marrows (p=0.007). CD34 + cells in MDS marrows expressed significantly higher levels of CD11c (p=0.007), CD117 (p=0.023) and CD33 (p=0.013) than their normal counterparts, with a trend towards lower expression of CD71 (p=0.053). Monocytes in MDS marrows expressed significantly lower levels of CD16 (p=0.006), CD36 (p=0.004) and CD117 (p=0.007) than their normal counterparts, with a trend towards lower expression of CD11c (p=0.059). We did not observe any differences in the antigen expression of MDS erythroblasts in comparison to their normal counterparts. A diagnostic score (0–7) was constructed taking into account the following seven parameters: expression of CD33 on granulocytes; CD11c, CD33 and CD117 on CD34+ cells; CD16, CD36 and CD117 on monocytes. This score distinguished all cases of MDS samples from controls. These results suggest that analysis of cell populations other than blasts could be useful in patients with MDS and that an immunophenotypic panel composed of markers commonly used in the diagnosis of acute leukemias could constitute a useful tool, in complement to morphological studies, for the diagnosis of myelodysplastic syndromes.
We report a case of B-cell chronic lymphocytic leukemia (B-CLL) with aberrant expression of the T-cell-associated antigen CD8, as revealed by two-color flow-cytometric analysis. DNA studies showed ...immunoglobulin heavy-chain gene rearrangement, but not of gamma-chain T-cell receptor, confirming the B-cell origin of the neoplastic cells. Ploidy analysis showed a tetraploid population and high S-phase fraction. B-CLL cells also carried trisomy 12, detected by fluorescence in situ hybridization. The identification of more cases with the same features would be necessary to establish the prognosis of this subtype of B-CLL.
In order to establish the response and tolerance to the intraarterial association of cis-platinum and bleomycin, we have treated 38 patients with advanced head and neck cancer with the following ...dosages: continuous infusion of bleomycin, 20 mg/m2/day on days 1 and 2, and cis-platinum, 100 mg/m2 in a 3-hr infusion on day 3. Each treatment cycle was repeated every 21 days, the duration being conditioned to tolerance and response. All the patients underwent at least one complete series of treatment. The results were as follows: 11 patients (29%) had complete remission, and 22 (58%) had partial remission. No instances were ascertained of local toxicity (stomatitis, dermatitis). Except for 2 patients with reversible facial paralysis and 6 with anemia, no other signs of general toxicity were ascertained. In conclusion, the intraarterial combination of cis-platinum/bleomycin has proved highly effective (87% response) whereas the low index of local and general toxicity renders the drugs suitable for use before surgery and/or radiotherapy.
Proteins from human parotid saliva were separated by use of a standardized technique of electrophoresis on polyacrylamide gel. Several fractions were eluted from the gel and studied by immunochemical ...methods (immunodiffusion, hemagglutination, and immunoelectrophoresis). The presence of IgA was demonstrated in the three slowest bands of the pattern.
