We have characterized vasoactive intestinal peptide (VIP) receptor/G-protein coupling in rat alveolar macrophage (AM) membranes and find that pertussis toxin treatment and antisera against Gαi3 and Gαs reduce high-affinity 125I-VIP binding, indicating that both Gαs and Gαi3 couple to the VIP-receptor. The predominant VIP-receptor subtype in AM is VPAC1 and we examined the G-protein interactions of the human VPAC1 that had been transfected into HEK293 cells. VPAC1 has a molecular mass of 56 kDa; GTP analogs reduced 125I-VIP binding to this protein demonstrating that high-affinity binding of VIP to the receptor requires coupling to G-protein. Functional VIP/VPAC1/G-protein complexes were captured by covalent cross-linking and analyzed by Western blotting. The transfected human VPAC1 receptor in HEK293 was found to be coupled to Gαs but not Gαi or Gαq. Furthermore, pertussis toxin treatment had no effect on VPAC1/G-protein coupling in these cells. These observations suggest that the G-proteins activated by VPAC1 may be dependent upon species and cell type.

It is demonstrated that methotrexate/cisplatin-sensitive L1210 cells express low levels of major histocompatibility complex (MHC) class II relative to the high levels expressed on methotrexate (MTX)/cisplatin-resistant L1210/DDP cells. L1210 cells express cell-surface Fas, while the L1210/DDP cells express no cell-surface Fas. Expression of costimulatory molecules B7-1/B7-2 and Fas is increased on L1210 cells, but not L1210/DDP, in the presence of methotrexate or trimetrexate (TMTX). Therefore, a component of the mechanism of action of some anti-cancer agents may be to facilitate immune recognition and T cell-directed, Fas-induced cell death. Loss of cell-surface Fas expression and failure of Fas (CD95)-dependent apoptotic death has been observed when cells develop drug resistance. The defect in apoptosis can be overcome by anti-cancer agents or experimental manipulation that induce Fas expression on the drug-resistant cells.

This study investigated potential mechanisms of oxidant resistance in alveolar macrophages (AM) isolated from Lewis rats exposed repeatedly to cadmium aerosols. Macrophages from Cd-adapted animals exhibited significantly greater resistance to oxidant-induced cytotoxicity than control cells when challenged with hydrogen peroxide in vitro. Elevations in glutathione peroxidase and glutathione reductase activities were associated with increased oxidant tolerance but catalase activity was unchanged. Metallothionein (MT) expression (protein and mRNA) was dramatically up-regulated in response to in vivo Cd exposure. A study using immunocytochemistry and in situ hybridization techniques revealed significant heterogeneity in the expression of metallothionein by AMs. The percentage of AMs positive for MT (protein and mRNA) and the degree of MT expression within individual cells increased in response to additional Cd exposures. A putative state of activation was suggested by the differences in size and number of inclusion bodies in macrophages from Cd-adapted animals and by secretion of a cytokine with interleukin-1-like characteristics. In summary, AMs from Cd-adapted animals are distinguished from control cells with respect to: (1) increased oxidant resistance, (2) secretion of cytokines, (3) elevations in enzymes associated with glutathione metabolism, and (4) up-regulation in metallothionein expression.

Collateral resistance to cisplatin and methotrexate has been reported in several cell lines. A murine leukemia cell line (L1210/DDP) selected for cisplatin resistance also has been shown to be highly resistant to methotrexate. Of the mechanisms proposed for methotrexate resistance, only changes in methotrexate transport into the cells were found in an earlier report. Methotrexate enters mammalian cells via an active transport system. In the present study, we demonstrated that the transport into the cell may be impaired in the resistant cells due to altered tyrosine phosphorylation of a membrane protein with a molecular mass of 66 kDa. This alteration was manifested by altered tyrosine phosphorylation of the 66 kDa protein and may be an underlying modification that renders the cells resistant to methotrexate. These results suggest involvement of tyrosine phosphorylation in folate transport and methotrexate resistance in L1210/DDP cells.

Synthetic procedures have been developed which lead to the 2-aza congeners 3 and several related N-oxides 4. The analogues 3 exhibited a wide range of in vitro cytotoxicity against L1210 leukemia, the human colon adenocarcinoma cell line LoVo, and the doxorubicin resistant LoVo/DX cell line. Selected analogues of 3 showed significant P388 antileukemic activity in mice with 3c exhibiting high activity. This activity was also retained in the related N-oxide 4a. These heterocyclic bioisosteric models are representative of the first anthracene-9,10-diones which display antileukemic activity comparable to mitoxantrone.

Isoprenylated proteins are believed to play an important role in DNA synthesis and subsequent cell cycle progression. Inhibition of the biosynthesis of these isoprenylated proteins results in a decrease in DNA synthesis and a characteristic G1 blockade of the cell cycle. This inhibition can be achieved by incubation of cells in the presence of a hydroxy-methylglutaryl-coenzyme A reductase inhibitor (lovastatin) and can be reversed by addition of exogenous mevalonate. When incubated in the presence of lovastatin, L1210 wild-type cells are not inhibited at G1 but rather progress to a G2/M blockade, which is reversible with exogenous mevalonate. Thymidine incorporation has also shown that DNA synthesis is occurring until this blockade is achieved. However, when L1210 cells resistant to cisplatin are incubated in the presence of lovastatin, the characteristic G1 cell cycle blockade and DNA synthesis inhibition occur. The understanding of this mechanism and the role that isoprenylated proteins play in the regulation of DNA synthesis and cell cycle progression may gain great insight into the abnormal control of the cancer cell.

The preparation and spectral properties of a series of complexes of general formula [PtCl(R′R″SO)(1,1- diaminomethylcyclohexane)]NO3 (R′R″SO=substituted sulfoxide) is reported. The complexes were studied for cytotoxicity and antitumor efficacy where high activity was seen for n-propyl- and n-butylsulfoxide derivatives. The results confirm previous reports that the biological activity of this series is dependent on the nature of the sulfur ligand and extends the type of useful sulfoxides to sterically hindered aliphatic ligands.

The complex (R,R-1,2-diaminocyclohexane)bis(shikimato)platinum(II) (shikimato = the anion of 3R,4S,5R-trihydroxy-1-cyclohexene-1-carboxylic acid), I, has been synthesized and purified by high performance liquid chromatography (HPLC). The complex is only moderately stable in aqueous solution. Its major hydrolysis product, also purified by HPLC, is proposed to be a unique complex type in which a single shikimate group is coordinated through both the carboxylate oxygen and the C(2) vinylic carbon of the shikimate moiety [Pt(R,R-dach)(O,C-shikimato)], II. In vitro, complex I is active against L1210 leukemia and against an L1210 cell line with acquired resistance to cisplatin. In vivo, the complex is active against L1210, P388, and B16 melanoma; this activity is highly schedule-dependent. Complex II is also active against L1210 leukemia.

The in vitro cytotoxicity and in vivo antitumor activity of bis(platinum) complexes of general formula [(PtX2-(L))2H2N(CH2)nNH2] (L = NH3, X = Cl or X2 = malonato or where L = py, X = Cl) is reported. Chloride complexes [(PtCl2(NH3]2H2N(CH2)nNH2] may exist as three possible isomers: those containing both coordination units in the cis configuration (2,2/c,c), both coordination units in the trans configuration (2,2/t,t), and the mixed cis,trans species (2,2/c,t), whose synthesis is reported here. The preparation of further complexes with sterically hindered diamine backbones, such as 2,5-dimethyl-2,5-hexanediamine, is exemplified. The biological activity of all complexes were compared. The 2,2/c,c complexes are particularly active in tissue culture in cells resistant both to cisplatin and its 1,2-diaminocyclohexane (dach) analogue. The inhibition of DNA synthesis in L1210/0 cells by the 2,2/c,c complexes is equivalent to that of cisplatin. The presence of at least one cis-[Pt(amine)2] unit appears necessary for activity in cell lines sensitive to cisplatin.

AbstractThe diamine(sulfoxide)‐Pt(II) complexes (III) and analogues of (R,R)and (S,S)‐trans‐1,2‐diaminocyclohexane are prepared as shown for (III) (no yields given).

We report experiments which test the toxicity of a new potential therapeutic agent, agarose-bound adriamycin (ImA). In C57Bl/6N mice this preparation is almost completely devoid of untoward effects when administered intraperitoneally; ImA lacks all the usual toxic repercussions of free adriamycin including abdominal adhesions, inflammatory peritonitis, weight loss and cardiotoxicity. The immobilized adriamycin is also inactive in a fetal mouse heart model of cardiac toxicity. This lack of toxicity is not due to an intrinsic inactivity of the drug, however, since previous studies have shown that polymer-bound adriamycin can kill actively dividing cells. We also show here that the immobilized drug can undergo redox reactions and interact with enzymes from isolated respiratory chain preparations, so the lack of cardiac toxicity in vivo is most likely due to inaccessibility of the target. These results suggest that polymer immobilized adriamycin lacks the toxicity of the parent compound and may present a useful approach to regional chemotherapy.

Two previously reported water soluble 1,2-diaminocyclohexaneplatinum(II) antitumor complexes with nitrogen-containing dicarboxylato ligands (N-substituted iminodiacetato(1,2-diaminocyclohexane)platinum(II) and aminomalonato(1,2-diaminocyclohexane)platinum(II)) were discovered to have significant residual impurities in the chemical formulations. Upon further purification each complex was found to be significantly less active in biological systems than previously reported. Each complex is stable in aqueous solution. This experience suggests that commonly accepted criteria for chemical identification and purity are inadequate for this type of complex. We hypothesize that tridentate bonding between the nitrogen-containing dicarboxylato group and platinum renders these complexes chemically stable and biologically inert.

Resistance to antineoplastic agents is the major obstacle to curative therapy of cancer. Tumor cell lines with acquired resistance to the antineoplastic agent cis -diamminedichloroplatinum(II) overexpressed metallothionein and demonstrated cross-resistance to alkylating agents such as chlorambucil and melphalan. Human carcinoma cells that maintained high levels of metallothionein because of chronic exposure to heavy metals were resistant to cis -diamminedichloroplatinum(II), melphalan, and chlorambucil. Furthermore, cells transfected with bovine papilloma virus expression vectors containing DNA encoding human metallothionein-II A were resistant to cis -diamminedichloroplatinum(II), melphalan, and chlorambucil but not to 5-fluorouracil or vincristine. Thus, overexpression of metallothionein represents one mechanism of resistance to a subset of clinically important anticancer drugs.