4'-Iodo-4'-deoxydoxorubicin disrupts the fibrillar structure of transthyretin amyloid.

Transthyretin (TTR) is a tetrameric protein synthesized mainly by the liver and the choroid plexus, from where it is secreted into the plasma and the cerebrospinal fluid, respectively. Some forms of polyneuropathy, vitreopathy, and cardiomyopathy are caused by the deposition of normal and/or mutant TTR molecules in the form of amyloid fibrils. Familial amyloidotic polyneuropathy is the most common form of TTR amyloidosis related to the V30M variant. It is still unclear the process by which soluble proteins deposit as amyloid. The treatment of amyloid-related disorders might attempt the stabilization of the soluble protein precursor to retard or inhibit its deposition as amyloid; or aim at the resorption of the deposited amyloid. The anthracycline 4'-iodo-4'-deoxydoxorubicin (I-DOX) has been shown to reduce the amyloid load in immunoglobulin light-chain amyloidosis. We investigated 1) whether I-DOX has affinity for TTR amyloid in tissues, 2) determined the I-DOX binding constants to TTR synthetic fibrils, and 3) determined the nature of the effect of I-DOX on TTR fibrils. We report that 1) I-DOX co-localizes with amyloid deposits in tissue sections of patients with familial amyloidotic polyneuropathy; 2) I-DOX strongly interacts with TTR amyloid fibrils and presents two binding sites with k(d) of 1.5 x 10(-11) mol/L and 5.6 x 10(-10) mol/L, respectively; and 3) I-DOX disrupts the fibrillar structure of TTR amyloid into amorphous material, as assessed by electron microscopy but does not solubilize the fibrils as confirmed by filter assays. These data support the hypothesis that I-DOX and less toxic derivatives can prove efficient in the treatment of TTR-related amyloidosis.

Delivery of methoxymorpholinyl doxorubicin by interleukin 2-activated NK cells: effect in mice bearing hepatic metastases.

The possibility of using interleukin 2 (IL-2)-activated natural killer cells (A-NK) to carry methoxymorpholinyl doxorubicin (MMDX; PNU 152243) to liver-infiltrating tumours was explored in mice bearing 2-day established M5076 reticulum cell sarcoma hepatic metastases. In vitro, MMDX was 5.5-fold more potent than doxorubicin against M5076 tumour cells. MMDX uptake by A-NK cells correlated linearly with drug concentration in the incubation medium [correlation coefficient (r) = 0.999]; furthermore, as MMDX incorporation was readily reproducible in different experiments, the amount of drug delivered by A-NK cells could be modulated. In vivo experiments showed that intravenous (i.v.) injection of MMDX-loaded A-NK cells exerted a greater therapeutic effect than equivalent or even higher doses of free drug. The increase in lifespan (ILS) following A-NK cell delivery of 53 microg kg(-1) MMDX, a dosage that is ineffective when administered in free form, was similar to that observed in response to 92 microg kg(-1) free drug, a dosage close to the 10% lethal dose (ILS 42% vs. 38% respectively). These results correlated with pharmacokinetic studies showing that MMDX encapsulation in A-NK cells strongly modifies its organ distribution and targets it to tissues in which IL-2 activated lymphocytes are preferentially entrapped after i.v. injection.

Uncoupling signal transducers from oncogenic MET mutants abrogates cell transformation and inhibits invasive growth.

The assumption that genes encoding tyrosine kinase receptors could play a role in human cancers has been confirmed by the identification of oncogenic mutations in the kinase domain of RET and KIT. Recently, homologous residues were found mutated in MET, in papillary renal carcinomas (PRCs). The link coupling these genetic lesions to cellular transformation is still unclear. METPRC mutations result in increased kinase activity and-in some instances, i.e., M1250T substitution-in changes in substrate specificity. A direct correlation occurs between the transforming potential of METPRC mutants and their ability to constitutively associate with signal transducers through two phosphorylated tyrosines (Y1349VHVNATY1356VNV) located in the receptor tail. Substitution of these "docking tyrosines" with phenylalanines leaves unaffected the altered properties of the kinase but abrogates transformation and invasiveness in vitro. Uncoupling the receptor from signal transducers with a tyrosine-phosphorylated peptide derivative (YpVNV) inhibits invasive growth induced by METPRC mutants. These data indicate that constitutive receptor coupling to downstream signal transducers is a key mechanism in neoplastic transformation driven by mutated MET and suggest a therapeutic strategy to target neoplastic diseases associated with this oncogene.

Adoptive transfer of lymphokine-activated killer cells loaded with 4'-deoxy-4'-iododoxorubicin: therapeutic effect in mice bearing lung metastases.

We studied the potential use of lymphokine-activated killer (LAK) cells loaded with 4'-deoxy-4'-iododoxorubicin (IDX) in adoptive immunotherapy experiments. Because LAK cells preferentially locate in the lung, we evaluated the therapeutic effect of IDX-loaded LAK cells in mice bearing lung metastases induced by B16F1 tumor cell injection. In vitro studies showed that LAK cells rapidly incorporated IDX, with maximum uptake at 15 min, followed by a plateau; drug efflux was initially rapid and then continued at a much slower rate. Evaluation of LAK cell cytotoxic activity against relevant target cells showed a 30% decrease after IDX treatment that progressed with time over the next 6 h. P388 tumor cell growth was inhibited by coculture with IDX-loaded LAK cells, thus demonstrating that the released IDX maintained its pharmacological activity. Finally, high performance liquid chromatography analysis of tissue IDX concentration revealed a considerably higher and long-lasting concentration in the lungs of mice receiving IDX-loaded LAK cells, compared to mice given injections of a comparable amount of free drug. Moreover, adoptive transfer of IDX-loaded LAK cells into tumor-bearing mice caused a significant reduction in the number of lung metastases versus control mice given injections of even higher doses of free drug.

Reversal of multidrug resistance by new dihydropyridines with low calcium antagonist activity.

The clinical use of Ca++ antagonist agents as modulators of multidrug resistance is limited by their strong vasodilator activity. This study reports data obtained by testing a series of new 1,4 dihydropyridine derivatives (DHPs) for their in vitro resistance modulating activity and their Ca++ antagonist effect. All the tested DHPs are active to increase doxorubicin activity with dose modifying factor values ranging between 2 and 47 on P388/DX cells and 12 and 36 on LoVo/DX cells. Their resistance modulating action is exerted through an increase of DX intracellular level. The Ca++ antagonist activity of DHPs, evaluated as capacity to inhibit the KCl-induced contractions in isolated Guinea pig ileum strips, is not related to their resistance modulating activity. This finding makes it possible to select, for further in vivo evaluations, compounds IX, X and XI, which have strong ability to overcome multidrug resistance and low Ca++ antagonist effect.

Establishment of L1210 leukemia cells resistant to the distamycin-A derivative (FCE 24517): characterization and cross-resistance studies.

N-deformyl-N-[4-N,N-bis(2-chloroethylamino)benzoyl] distamycin-A (FCE 24517) is a new cytotoxic anti-tumor agent in phase-1 clinical trials. We have isolated stable FCE-24517-resistant cell sublines from murine leukemia L1210 cells by in vitro exposure to the drug. FCE 24517 selects a mixed population of resistant cells: the L1210/24517(1) cell line in vitro was in fact resistant to the selecting agent (RI 48.3), as well as to L-PAM (RI 5.4) and DX (RI 8.6) and over-expressed the mdr-I gene. When L1210/24517(1) cells were implanted in vivo and evaluated for sensitivity to the same agents, resistance was observed only to FCE 24517 and partially to L-PAM, whereas DX had the same anti-tumor efficacy as on the sensitive line. The clone derived from the above subline (L1210/24517(2)) was resistant to FCE 24517, distamycin-A and other cytotoxic compounds bearing the distamycin-A skeleton, and fully sensitive to DX and other anti-tumor compounds involved in the multi-drug resistance mechanisms, with a complete disappearance of the mdr phenotype. L1210/24517(2) cell line is partially cross-resistant to L-PAM, this resistance being accounted for by higher GSH intracellular levels, which however do not influence the resistance to FCE 24517. In fact, BSO treatment was capable of significantly modifying only the cytotoxicity of L-PAM. Our data suggest that L1210/24517(2) cells present a mechanism of resistance specific for FCE 24517 and related molecules.

Biological profile of FCE 24517, a novel benzoyl mustard analogue of distamycin A.

FCE 24157 (chemically (beta-[1-methyl-4-(1-methyl-4--[1-methyl-4-(4-N,N- bis(2-chloroethyl) amino-benzene-1-carboxy-amido) pyrrole-2-carboxiamido]pyrrole-2-carboxyamido)pyrrole-2-c arboxyamido]) propionamidine, hydrochloride) is a distamycin A (Dista A) derivative bearing a benzoyl mustard moiety instead of the formyl group at the N-terminal. Contrary to Dista A, FCE 24517 has been found to display potent cytotoxic activity on human and murine tumour cell lines. The compound maintains activity on melphalan (L-PAM)-resistant cells, whereas cross-resistance is observed on doxorubicin-(DX)-resistant cells. In vivo, FCE 24517 was found to possess evident antineoplastic activity on a series of murine transplanted solid tumours and human tumour xenografts. The following neoplasms were in fact found to be sensitive to FCE 24517 treatment: M14 human melanoma xenograft, N592 human small cell lung carcinoma, MTV murine mammary carcinoma, Colon 38 murine carcinoma, PO2 murine pancreatic carcinoma and M5076 murine reticulosarcoma. Lower effectiveness was observed against the murine P388 and Gross leukaemia, Lewis lung murine carcinoma, LoVo human colon carcinoma xenografts and A459 human lung adenocarcinoma. Against the murine L1210 leukaemia, FCE 24517 displayed a clear activity only when the tumour was transplanted i.p. and treatment was given i.p., whereas only marginal activity was seen against this leukaemia if transplanted i.v. and the drug was given i.v. As true also in vitro, FCE 24517 was effective against i.p. implanted L1210 leukaemia resistant to L-PAM. The mode(s) of action of this new compound is under active investigation.

Selective DNA interaction of the novel distamycin derivative FCE 24517.

N-Deformyl-N-(4-N-N,N-bis(2-chloroethylamino)benzoyl)distamy cin A (FCE 24517) is a novel cytotoxic and antitumor agent shortly to be investigated in phase I clinical trials. It was equally effective in inhibiting the growth of the murine L1210 line and of a subline (L1210/PAM) resistant to nitrogen mustards, whereas distamycin A was virtually inactive. The cellular uptake and retention of FCE 24517 and distamycin A were similar, thus excluding the possibility that this marked variation in cytotoxic activity was due to different intracellular concentrations of the two compounds. FCE 24517 did not appear to act as an inhibitor of macromolecule synthesis. As shown by radioactively labeled precursor incorporation only 24 h after drug treatment a significant inhibition of DNA synthesis was observed in L1210 or in L1210/PAM, when a marked proportion of cells was arrested in premitotic phase. FCE 24517 did not cause DNA breaks, DNA interstrand cross-links, or DNA-protein cross-links in L1210 cells exposed to active drug concentrations. A very low amount of radioactivity was found to be bound irreversibly to DNA in L1210 cells exposed for 1 h to [14C]FCE 24517. Using plasmid pBr322 DNA fragments in a modified version of the Maxam and Gilbert DNA sequencing technique we found no detectable binding of FCE 24517 to N-7-guanine (the major site of alkylation for classical alkylating agents), whereas some alkylations to adenine (presumably to N-3-adenine) were demonstrated. Thus it appears that FCE 24517 is a novel antitumor agent with a mode of action different from that of the drugs currently used in the clinic. In summary it is suggested that FCE 24517 acts by causing a few selective alkylations to adenines in the minor groove of DNA, although the precise base sequence necessary has yet to be elucidated.

Modulation of multidrug resistance by verapamil or mdr1 anti-sense oligodeoxynucleotide does not change the high susceptibility to lymphokine-activated killers in mdr-resistant human carcinoma (LoVo) line.

Two sublines were derived from the colon adenocarcinoma line LoVo, the first one was sensitive (LoVo/H) and the second one was made resistant to doxorubicin (LoVo/Dx). When tested for susceptibility to lysis by different types of immune effectors, LoVo/Dx appeared more sensitive than LoVo/H to the killing of CD3+CD5+CD16-, CD3- CD16+)-enriched lymphokine activated killers (LAK) or activated macrophages. In order to check whether this effect was due to different expression of glycoprotein P170 between the two LoVo sublines (30% vs. 90% of positive cells), a pharmacological and genetic modulation of P170 was carried out in LoVo cells. Treatment of LoVo/Dx with the calcium channel blocker verpamil (VRP), strongly impaired P170 function as evaluated by reduced Dx resistance, without affecting the lysability of LoVo/Dx cells by LAKs. Moreover, the significant inhibition of P170 expression resulting from the treatment of LoVo/Dx with mdr1 anti-sense olideoxynucleotide also failed to change the high lysability of LoVo/Dx by LAKs. These results, therefore, indicate that molecules other than P170 are involved in the increased lysis of LoVo/Dx subline by immune effectors and that down-regulation of the P170 expression or function will not reduce the potential effectiveness of cancer chemo-immunotherapy.

Pharmacology and clinical toxicity of 4'-iodo-4'-deoxydoxorubicin: an example of successful application of pharmacokinetics to dose escalation in phase I trials.

In a prospective phase I trial involving 35 patients with metastatic carcinoma, we tested a pharmacokinetic strategy for guiding dose escalation of the anthracycline 4'-iodo-4'-deoxydoxorubicin (I-DOX), a new analogue reported to be more potent and less toxic than doxorubicin. This strategy is potentially a safe and more rapid way of determining the maximum tolerated dose (MTD) of anticancer agents. Retrospective studies have shown that the total plasma drug exposure after a dose lethal to 10% of mice (LD10) is approximately equivalent to the total exposure produced in humans by the MTD. Thus, we intended to aim dose escalation in humans to achieve the area under the curve for I-DOX plasma concentration x time (AUC) equivalent to that produced in mice by an LD10. However, differences in I-DOX pharmacokinetics and metabolism in BDF1 mice and humans at the initial dose prevented immediate application of this strategy. Therefore, we escalated the dose by the modified Fibonacci scheme while investigating the pharmacology of I-DOX and its major plasma metabolite 4'-iodo-4'-deoxy-13-dihydrodoxorubicin (I-DOXOL). Plasma pharmacokinetics was characterized by rapid elimination and extensive metabolism of I-DOX to I-DOXOL. The ratio of I-DOXOL to I-DOX plasma AUC was 12.8 +/- 7.3 SD. The plasma pharmacokinetics of I-DOX and I-DOXOL were linear in the range of tested doses (2-90 mg/m2). The LD10 in mice was 6.8 mg/kg for I-DOXOL and 6 mg/kg for I-DOX, and the concentration of drug that inhibited by 50% (IC50) the growth of human granulocyte-macrophage colony-forming units (CFU-GM) was 80 nM for I-DOXOL and 50 nM for I-DOX. From these findings, we concluded that the toxic effects of I-DOX and I-DOXOL are equivalent and reset the pharmacokinetic target of escalation to the sum of I-DOX and I-DOXOL AUCs at I-DOX LD10. Then we safely applied pharmacokinetically guided escalation to determine the MTD (80 mg/m2). The plasma AUC of I-DOX and I-DOXOL at the human MTD is 71% of the AUC at mouse LD10. The only dose-limiting toxic effect was severe granulocytopenia.

Ultrastructural cytoskeleton alterations and modification of actin expression in the NIH/3T3 cell line after transformation with Ha-ras-activated oncogene.

Cytoskeleton alterations of NIH/3T3 fibroblast monolayers transfected with Ha-ras-activated oncogene were studied by immunofluorescence, immunoelectron microscopy, and immunoelectrophoretic analysis of actin isoforms. Transformation foci were found to consist of cells with a round shape and rare stress fibers that spread sparsely, forming rare focal contacts and fibronexuses. The loss of stress fibers in transformed cells was confirmed by staining with rhodamine-phalloidin and with a fluorescinated anti-non-muscle cell actin antibody. The transformed cells were anchored to the substrate prominently by filaments that contained fibronectin, as showed by immunoelectron microscopy. A down-regulation of alpha-actin isoform was observed by immunofluorescence and immunoblotting analysis using a specific monoclonal antibody. The diffuse distribution of alpha-actin, lacking a specific association with stress fibers, challenges the hypothesis of a connection between alpha-actin down-regulation and stress fiber loss.

Characterization of papillomavirus polypeptides from bovine cutaneous fibropapillomas.

Virions of bovine papilloma virus (BPV) were isolated from a pool of cutaneous bovine fibropapillomas and purified by CsCl gradient centrifugation. SDS-PAGE revealed several polypeptides with an Mr ranging from 76K to 19K. Western blot analysis of the viral isolate identified additional polypeptides when a rabbit anti-BPV serum was used, but only the main capsid component of 57K when a rabbit antiserum raised against human papillomavirus was used. The viral preparation was then 125I-labelled and further purified by gel filtration. SDS-PAGE of immunoprecipitates of the anti-BPV serum with different fractions from the chromatographic column revealed the polypeptides of 76K, 57K and 28K to be viral structural components. The 28K polypeptide, not previously characterized, was shown to be composed of several molecular forms, migrating over a pH range of 3.5 to 4.6 when analysed by two-dimensional PAGE. Following SDS-PAGE performed under non-reducing conditions, the 28K and 76K polypeptides and the main capsid component of 57K appeared to be linked by disulphide bridges to form hetero- or homopolymers.

Early lymphocyte activation molecule defined by the monoclonal antibody MLR-3: biochemical and functional studies.

The MLR-3 monoclonal antibody reacts with activated but not with resting lymphocytes. We report that MLR-3 identifies an early activation molecule since its binding is detectable on T cells 1.5-2 hr after in vitro activation. Its expression, therefore, does not require DNA synthesis and precedes, by many hours, that of the receptors for interleukin-2 (IL-2R) and transferrin (TF-R). The MLR-3 antigen is also found on activated thymocytes (including the large early thymic CD3- subset) and B cells. The majority of T- and B-lymphoblastoid cell lines, as well as the myeloid and erythroid cell lines HL60, GM1 and K562, are MLR-3+; conversely, non-haemopoietic cell lines are MLR-3 negative. Seventy percent of B-cell chronic lymphocytic leukaemia and 15% of B non-Hodgkin's lymphomas (B-NHL) are MLR-3+. On tissue sections MLR-3 is reactive with epithelia, sweat glands, hair follicles and Henle's loop but not with vessels, connective, endothelium and many other tissues. In vitro studies show that MLR-3 (1-100 micrograms/ml) significantly alters the thymidine uptake of mitogen-treated lymphocytes:augmentation is found when T and B cells are induced with TPA-Ionomycin and reduction when induced with phytohaemoagglutinin (PHA) or Staphylococcus aureus Cowan strain 1 (SAC), respectively. On SDS-PAGE, MLR-3 immunoprecipitates a disulphide-linked heterodimer of MW 29,000-35,000: both subunits are glycosylated, phosphorylated and exhibit a pI of 4.1 and 5.0, respectively. Our data, particularly the in vitro results, suggest that the MRL-3 molecule could have an important role in the early hours of activation for the progression of resting lymphocytes into mitosis.

Monoclonal antibodies against NIH 3T3 cells transformed by human thyroid carcinoma DNA.

First cycle transformants of NIH 3T3 cells transfected with metastatic human thyroid carcinoma DNA were used as immunogen to obtain monoclonal antibodies (MAbs) against normal and transformation-related antigens. The transformed cell line (M33) was shown to contain Alu sequences. Two MAbs were selected on the basis of their differential reactivity toward untreated NIH 3T3 cells or the transformed M33 cell line. By immunofluorescence, immunoelectronmicroscopy and biochemical analysis, the first MAb (MTr1) was demonstrated to recognize an epitope on cytoskeletal filaments of proliferating murine fibroblasts. Similar MTr1-labelled filaments were also found to accumulate into cytoplast-like structures spontaneously produced by M33 cells. The characterization by immunofluorescence of MTr2, the second MAb, indicates that it recognizes a specific human antigen associated with normal thyroid epithelial cells and differentiated thyroid tumors.

Ly-5.185 molecule is associated with thymic maturation of lymphocytes but not with their cytotoxic activity.

The Ly-5 system is a composite family of molecules with different molecular weight. Each molecular form is characteristic of a different lymphoid population, in particular a 175,000- to 185,000-dalton doublet is peculiar of thymocytes and peripheral T cells. Athymic nude mice, however, lack the 185,000-dalton form on their lymphocytes. To investigate whether the expression of the 185,000 daltons requires the presence of thymus, we induced nude mice lymphocytes to differentiate into mature T cells in vivo by thymus grafting and, in vitro, into T cytotoxic cells (CTL) by cocultivation with alloantigen plus recombinant interleukin 2 (IL-2). The appearance of the 185,000-dalton form on lymphocytes of thymus-grafted nude mice but not on in vitro activated cytotoxic cells indicates an active role of thymus in the induction of this molecule and suggests that 185,000-dalton form could be a marker of intrathymic differentiation but not of the cytotoxic activity of T cells.

DBA/2-like minor histocompatibility antigens on a BALB/c lymphoma. A BALB/c anti-DBA/2 serum which lyses the tumor and blocks BALB/c anti-tumor and anti-DBA/2 effectors.

We have previously shown that BALB/c anti-DBA/2 T cells can lyse the Moloney virus-induced BALB/c lymphoma YC8. In order to determine whether serologically defined minor histocompatibility antigens (MiHA) cross-reacting with those of DBA/2 tissues are present on YC8, we produced an antiserum directed against non-H-2 antigens by immunizing BALB/c mice with DBA/2 Con A and lipopolysaccharide (LPS)-induced lymphoblasts. In a direct complement-dependent cytotoxicity assay, the antiserum (OR-1) lysed DBA/2 and YC8 but not BALB/c lymphocytes and blasts. No reactions against viral antigens were detected in the antisera as shown by the lack of cytotoxicity on a panel of lymphomas expressing a variety of viral antigens. In addition, OR-1 was able to specifically block a cytotoxic T lymphocyte (CTL), H-2-restricted BALB/c anti-DBA/2 cytotoxic response when bound to DBA/2 or to YC8 target cells. These results indicate that antigens cross-reacting between YC8 lymphoma and DBA/2 tissues are serologically defined MiHA of DBA/2 background and that OR-1 serum can block a CTL reaction by binding to target antigen rather than to major histocompatibility complex products.

Cell surface antigens of chemically induced fibrosarcomas: detection by a monoclonal antibody of a tumor-restricted Mr 12,000 protein gag antigen encoded by a dual-tropic murine leukemia virus.

Fusion products of spleen cells of W/FuDp rats immunized with a methylcholanthrene-induced BALB/c sarcoma, CA-2, and mouse myeloma cells were screened in an attempt to identify a monoclonal antibody defining the individually distinct tumor-specific transplantation antigen of CA-2. A hybridoma, MP/69/04, was isolated which produces an IgG2a monoclonal antibody that recognized a tumor-restricted antigen of CA-2. In direct binding assay, MP/69/04 reacted only with 2 of 15 methylcholanthrene induced BALB/c sarcomas tested. Thymus, spleen, lymph nodes, bone marrow, brain, adult lung fibroblasts, newborn muscle fibroblasts and 3T3 cells were negative. Absorption tests revealed, however, expression of the MP/69/04 determinant on 8 of the 12 murine leukemia virus (MuLV) producer BALB/c sarcoma tested. The antigen was not detected on any of the three non-producer sarcomas tested nor on a wide range of normal tissues and cell lines. An N-dualtropic MuLV was isolated from CA-2, and cell lines susceptible to infection by this virus were shown to express the MP/69/04 epitope. By Western blotting, the MP/69/04 epitope was identified as being expressed on the MuLV structural protein with a molecular weight of 12,000, present in CA-2 cells and in the purified CA-2 MuLV. These results indicate the MP/69/04 antigen is not a unique tumor-specific transplantation antigen but is a gag product of a recombinant retrovirus which is expressed on the cell surface of many MuLV + methylcholanthrene-induced BALB/c fibrosarcomas.