Antitumour activity of miltefosine alone and after combination with platinum complexes on MXT mouse mammary carcinoma models

Miltefosine, an alkylphosphocholine structurally related to alkyllysophospholipids showed highly selective antitumour activity against the hormone-sensitive variant of the s.c. transplantable MXT mouse mammary adenocarcinoma, the ovary-dependent MXT (M3.2), whereas it was inactive against the hormone-insensitive MXT (M3.2) OVEX variant. A dose of 32 mg/kg miltefosine p.o. daily for 5 weeks was well tolerated. Histopathological evaluation gave no signs of gastroenteral toxicity. After therapy the microarchitecture of the MXT (M3.2) tumours changed from that of a moderately differentiated adenocarcinoma to that of an anaplastic mammary carcinoma. A dose of 16 mg/kg miltefosine p.o. daily, though in effective per se, enhanced the antitumour activity of suboptimal i.p. doses of cisplatin and the hormone-like platinum analogue [meso-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]dichloroplatinum(II). Furthermore, it was shown, that miltefosine exhibited no (anti)hormonal properties. However, the mechanism of action of miltefosine remains unclear.     

Studies on high-dose chemotherapy of amygdalin in murine P388 lymphocytic leukaemia and P815 mast cell leukaemia

Summary The anti-tumor activity of amygdalin (NSC 251222), commercially known as Laetrile, was investigated using P388 lymphocytic leukaemia and P815 mast-cell leukaemia in BDF₁ mice. Doses varying from 200 mg/kg to 2,000 mg/kg were used following the days 1 and 5 and days 1,5 and 9 schedules. Despite treatment with high doses of amygdalin there was no prolongation in the life-span of mice bearing either P388 or P815 tumor.     

Different response of murine and human mammary tumour models to a series of diastereoisomeric [1,2-bis(difluorophenyl) ethylenediamine]dichloroplatinum(II) complexes

Summary A series of isomeric [1,2-bis(difluorophenyl) ethylenediamine]dichloroplatinum(II) complexes and cisplatin were tested on the P388 leukemia and on the murine hormone-independent MXT (M3.2) OVEX and the ovarian — hormone — dependent MXT (M3.2) mammary carcinoma for evaluating antineoplastic activity against breast cancer in vivo. Although these results were heterogeneous, a trend to the 2,6-difiuorosubstituted compound as the most active platinum complex was observed. For the development of a large-scale in vitro screening method on human breast cancer cell lines, cell number, [³H]thymidine incorporation, and crystal violet staining were evaluated as parameters for end-point determination. Chemosensitivity testing on the human breast cancer cell lines MDA-MB-231 and MCF-7 unam-biguously identified [1,2-bis(2,4-difluorophenyl)ethyle-nediamine]dichloroplatinum(II) as the complex with the highest activity in the crystal violet micro-assay. In equimolar concentration this compound was superior to cisplatin on both cell lines. The analysis of the conflicting results of this study indicates that murine mammary carcinomas are most probably unrealistic and inappropriate models for the screening of cytotoxic platinum complexes with potential activity on human breast cancer.Abbreviations PBS phosphate-buffered saline - PEG polyethlyeneglycol Dedicated to Professor Franz Lux on the occasion of his 65th birthday     

Characterization of tumour cells in concomitant chronic lymphocytic leukaemia and acute monocytic leukaemia.

A 68-year-old woman presented with haematological changes of chronic lymphocytic leukaemia and acute monocytic leukaemia. This diagnosis was confirmed by identification of cell surface markers for T and B lymphocytes and the identification of abnormal immunoglobulins and lysozyme in serum and urine.     

Murine models of chronic lymphocytic leukaemia: role of microRNA-16 in the New Zealand Black mouse model

Mouse models are valuable tools in the study of human chronic lymphocytic leukaemia (CLL). The New Zealand Black (NZB) strain is a naturally occurring model of late-onset CLL characterized by B-cell hyperproliferation and autoimmunity early in life, followed by progression to CLL. Other genetically engineered models of CLL that have been developed include (NZB x NZW) F1 mice engineered to express IL5, mice expressing human TCL1A, and mice overexpressing both BCL2 and a tumour necrosis factor receptor-associated factor. The applicability to human CLL varies with each model, suggesting that CLL is a multifactorial disease. Our work with the de novo NZB model has revealed many similarities to the human situation, particularly familial CLL. In NZB, the malignant clones express CD5, zap-70, and have chromosomal instability and germline Ig sequence. We also identified a point mutation in the 3'-flanking sequence of Mirn16-1, which resulted in decreased levels of the microRNA, miR-16 in lymphoid tissue. Exogenous restoration of miR-16 to an NZB malignant B-1 cell line resulted in cell cycle alterations, suggesting that the altered expression of Mirn15a/16-1 is an important molecular lesion in CLL. Future studies utilizing the NZB mouse could ascertain the role of environmental triggers, such as low dose radiation and organic chemicals in the augmentation of a pre-existing propensity to develop CLL.     

Overadditive synergism between the intercalators mitoxantrone and lucanthone in advanced L 1210 and P 388 leukemia

Summary The combination of mitoxantrone with lucanthone, a schistosomicidal and nonmyelotoxic agent, yielded a therapeutic synergism in L 1210 and P 388 leukemia with no increase in toxicity. In that combination the nonmyelotoxic lucanthone enabled the use of the optimal dose of mitoxantrone. The recent hypothesis that planar polycyclic aromatic compounds, mostly comprised by the term intercalators, intercalate with DNA or bind to DNA may need receiving with respect to membrane target sites.Dedicated to Professor Dietrich Schmähl on the occasion of his 60th birthday     

DNA ploidy level assessments in 83 human brain metastases relationship to the survival of 35 patients

The nuclear DNA content (NDA ploidy) level was determined in a series of 83 human brain metastases, for which 35 complete clinical follow-ups were available. The DNA ploidy level determination was carried out by means of DNA histogram types. The results show that certain brain metastases were diploid, while others exhibited aneuploidy levels ranging from low to very high. The present study also shows that a significant proportion, i.e. 18%, of the 83 brain metastases, exhibited very high levels of aneuploidy, i.e. hypertetraploidy, hyperpentaploidy and octoploidy. We had previously observed that this feature appeared only rarely, i.e. in less than 2% of primary nervous tumours. Furthermore, the present study shows that DNA ploidy level in brain metastases is related significantly (P<0.001) to patient survival. Indeed, while 9/13 (69%) patients with diploid brain metastases survived longer than 9 months, none (0%) of the 22 patients with aneuploid brain metastases survived longer than the 9 months following the diagnosis of their brain metastases.The present work was carried out on the basis of grants awarded by the Fonds de la Recherche Scientifique Médicale (FRSM, Belgium).     

In vivo influence of androgens on the cell kinetics and chromatin pattern of the MXT mouse mammary tumor treated or not by aminogluthetimide

Summary We characterized the influence of androstenedione, 5--dihydrotestosterone and 17--estradiol on the chromatin organization and the cell kinetics (distribution of the cells into the G0-G1, S and G2+M fractions) of MXT mouse mammary tumors grafted onto animals that were left intact or that were oophorectomized and/or treated with aminogluthetimide. The cell kinetics and chromatin pattern were monitored by analyzing Feulgen-stained MXT imprint smears through a cell image processor, the SAMBA 200 system. We have also assayed the estrogen and progesterone receptors of these MXT tumors, which appeared to possess both of these biological markers. Our results show that ovariectomy or aminogluthetimide treatment slowed down the MXT tumor growth without any additive effect between them. Androstenedione exerted a stimulating influence on the cell kinetics, which were very similar to those observed after estradiol administration; the treatment of castrated animals with aminogluthetimide completely abolished this androstenedione-induced stimulating influence, however. This androgen lacked any apparent efficiency to transform cell nuclei from a state of castration-induced chromatin condensation into a thinly textured and decondensed state, as did estradiol. Dihydrotestosterone was able to activate the cell kinetics of MXT tumor grafted onto castrated animals as well as those of MXT neoplasms grafted on oophorectomized mice treated with aminogluthetimide. Dihydrotestosterone also possesses the property to transform condensed chromatin into decondensed chromatin. It thus appeared that androstenedione and dihydrotestosterone could activate MXT cell proliferation, as did estradiol, although it appeared that their mechanisms of action were quite distinct from each other.Abbreviations AD androstenedione - AG aminogluthetimide - DHT dihydrotestosterone - E2 17--estradiol - ITC intact - OO ovariectomized This work is supported by grants awarded by the I.R.S.I.A. and the Fonds de la Recherche Scientifique Médicale (FRSM), BelgiumThe authors are grantees of Institut pour l'encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture (IRSIA)     

Metabolism and binding of androgens by mouse mammary tumour cells in culture

Both testosterone and 5α-dihydrotestosterone (DHT) increased the growth rate of androgendependent mouse mammary tumour cells (S115) in culture. DHT was effective at lower concentrations and increased the thymidine labelling index with a shorter lag-period than testosterone. The S115 cells converted testosterone to DHT, 5α-androstane-3α-17β-diol, androsterone and androstanedione. DHT was not converted to testosterone. All this data was consistent with the view that testosterone exerted its biological effect in this system by conversion to DHT. However, the high affinity, 8–9 cytoplasmic receptor bound testosterone as well as DHT. Testosterone competed with DHT for binding sites and vice versa; oestradiol 17β blocked the binding of both androgens but no binding of ³H-oestradiol-17β to an 8–9 component was detected. After incubation of cells with ³H-testosterone at 37 °C a 5S receptor could be extracted from nuclei. The major radioactive steroid in the nucleus under these conditions was testosterone. This binding data suggests that testosterone might be biologically active without metabolic conversion to DHT.     

Flow cytometry and the study of central nervous disease in patients with acute leukaemia

Central nervous system (CNS) leukaemia is still a matter of debate and new technologies are required to improve the classic morphological definition. One hundred and sixty-eight cerebrospinal fluid (CSF) samples from 31 patients with acute leukaemia were analysed by flow cytometry and conventional cytology. Concordant positive and negative findings were found in 158 samples but 10 produced discrepant results. Cytology seemed to offer more precise information in one CSF sample and flow cytometric accuracy could be demonstrated in five samples. We conclude that flow cytometry is of great help in confirming CNS leukaemia and eliminating other conditions. Therefore, leukaemic patients can benefit from double cytological and flow cytometric CSF studies.     

Correction of Bcl-x splicing improves responses to imatinib in chronic myeloid leukaemia cells and mouse models

Imatinib mesylate (IM) resistance has become a major clinical problem for chronic myeloid leukaemia (CML). It is known that Bcl-x splicing is deregulated and is involved in multiple malignant cancer initiation and chemotherapy resistance, including CML. The aim of the present study was to correct the abnormal splicing of Bcl-x in CML and investigate the subsequent malignant phenotype changes, especially response to IM. The aberrant Bcl-x splicing in CML cells was effectively restored using vivo-Morpholino Antisense Oligomer (vMO). CCK-8 cell viability assay and flow cytometry showed that restoring of Bcl-x splicing increases IM-induced growth inhibition and apoptosis of K562 cells. Moreover, a more significant similar phenomenon was observed in imatinib-resistant CML cell lines K562/G01. Finally, establishment of CML xenograft model had also proved that correcting Bcl-x splicing in vivo can also enhance the anti-tumor effect of IM. Our findings suggest that vMO co-operating with IM can effectively increase the sensitivity of CML cells to IM both in vitro and in vivo, and Bcl-x splicing could become good candidates for chemotherapy-sensitized target in IM-resistant CML.     

Low expression of the putative tumour suppressor gene gravin in chronic myeloid leukaemia, myelodysplastic syndromes and acute myeloid leukaemia

The putative tumour suppressor gene gravin is down-regulated in several solid tumours and is implicated in tumorigenesis. We have evaluated the expression levels of the gravin gene in the CD34(+)/blast cells of a range of myeloid malignancies as compared with controls using real-time quantitative polymerase chain reaction (PCR). Gravin was markedly down-regulated in 41 of 41 patients with acute myeloid leukaemia (AML), nine of 10 patients with myelodysplastic syndromes (MDS) and 33 of 33 patients with chronic myeloid leukaemia (CML), of whom 24 were in blast crisis (BC). We have shown that gravin is consistently down-regulated in the CD34(+)/blast cells of myeloid malignancies and may play a role in the molecular pathogenesis of these disorders.     

Polymorphism of the tumour necrosis factor-alpha and lymphotoxin-alpha genes in chronic lymphocytic leukaemia

The neoplastic cells of CLL are able to produce TNF which is known to stimulate the proliferation of CLL cells in an autocrine and paracrine manner. Genetic polymorphism of molecules of the TNF ligand superfamily has been described and certain alleles were suspected to predispose to variant biological responses. Previously, the rare allele TNFB*1 of the TNF-β/lymphotoxin (LT)-α gene (NcoI, asparagine at amino acid position 26) was found to be associated with a stronger LT-α response of PBMC in vitro .
We now report on a significant increase of the allele TNF1 (TNFA −308 G) of the TNF-α promoter/enhancer polymorphism in a group of 73 CLL patients when compared to healthy individuals (RR=3.18, 95% confidence interval 1.57–8.3; P =0.006). The allelic distribution of the TNF-β/LT-α NcoI polymorphism did not differ significantly from randomized healthy controls. On the other hand, the frequency of the allele TNFB*2 was increased in CLL patients with advanced clinical stage ( P =0.0004). These findings indicate immunogenetic associations involving polymorphisms of cytokine genes serving as paracrine and autocrine growth factors, which thus can contribute to the pathogenesis of the TNF/LT-sensitive haematological malignancy CLL.     

Polymorphism of the tumour necrosis factor-alpha and lymphotoxin-alpha genes in hairy cell leukaemia

Hairy cell leukaemia (HCL) is a rare chronic B lymphoproliferative disorder which can lead to severe pancytopenia and several immunologic abnormalities. The pathogenetic role of tumour necrosis factor (TNF)-α in HCL prompted us to study the potential contribution of functionally important genetic polymorphisms of the TNF gene cluster in a large group of patients with HCL. The TNF-α (−308  bp) promoter/enhancer point mutation and two polymorphisms located within the first intron of the lymphotoxin (LT)-α gene showed neither significant allelic deviation for the patient group nor, after analysis of clinical characteristics such as blood counts, stable or progressive disease or response to therapy.     

Rapid and sensitive non-radioactive assay for the detection of clonal gene rearrangements in B-lineage acute leukaemia

Summary. We have applied a simple and non-radioactive assay system used in conjunction with polymerase chain reaction amplification and high-resolution polyacrylamide gel electrophoresis to detect clonal immunoglobulin gene rearrangements in leukaemia patient DNA. This technique is as sensitive as isotopic methods, allowing the detection of clonal DNA diluted 1 in 10 000 in non-clonal DNA.     

Comparison of flow cytometry and enzyme cytochemistry for the detection of myeloperoxydase in acute myeloid leukaemia: interests of a new positivity threshold

Following the recommendations of the European Group for the Immunological Characterization of Leukaemias (EGIL) in 1995, few reports have been published comparing enzyme cytochemistry (EC) and flow cytometry (FC) for the detection of myeloperoxydase (MPO) in acute myeloid leukaemia (AML). The EGIL guidelines defined MPO positivity in FC, by the presence of this enzyme in 10% or more of the blast cells. We studied 136 adult patients with the systematic use of both EC and FC, using a 3% threshold for positivity for EC, and 10% and 3% consecutively for FC. FC was less sensitive than EC using the currently recommended threshold of 10%, but a 3% cut-off showed more sensitivity and was superior to EC. The joint use of both techniques identified 14 discordant patients (positive in FC/negative in EC or vice versa), all of whom displayed at least one poor-prognosis biological factor, which correlated with a mediocre clinical result. In conclusion, we recommend that the cut-off for a positive MPO value should be lowered to 3%, and suggest that the concomitant use of FC and EC is a fast clinically relevant prognostic tool.     

Substance P analogs displace sigma binding differentially in the brain and spinal cord of the adult mouse

We have previously observed similarities in the behavioral effects produced by the NH₂-terminus of the undecapeptide substance P (SP) and by 1,3-di(2-tolyl)-guanidine (DTG) in the adult mouse. The present series of experiments indicate differences in the rank-order of potency of sigma ligands [DTG; haloperidol (HAL)], SP analogs [SP; SP(1-7); SP(5-11); [D-Pro², D-Phe⁷]-SP(1-7) (D-SP(1-7))] and miscellaneous compounds [morphine (MOR), naloxone (NAL)] at competing for [³H]-DTG binding sites in the mouse brain and spinal cordin vitro: Brain; DTG = HAL SP = MOR = NAL SP(1-7) D-SP(1-7) SP(5-11): Spinal cord; DTG = HAL SP(1-7) = MOR = NAL SP D-SP(1-7) = SP(5-11). The observed difference in the rank-order potencies of the displacing ligands at these same binding sites supports the notion of two distinct populations of sigma binding sites in these tissues in the adult mouse. Given the low (micromolar) potency of SP analogs at displacing [³H]-DTG binding in the present series of experiments, it is unlikely that the similar behavioral effects we have previously observed elicited by SP(1-7) and DTG in the adult mouse are a result of a direct action of SP(1-7) at the sigma binding site.