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Fluorescence-microscopic studies with dansylated estramustine (DnsEM) has permitted investigation of the mechanism of estramustine (EM) uptake in live human prostatic tumor cells (DU 145). DnsEM appeared to enter cells rapidly at the peripheral cell margins. A progressive increase in fluorescence was observed until the perinuclear material and cytoplasm were labeled brightly and the nucleoplasm was labeled faintly. Light microscopy showed that DnsEM is assimilated first in preexisting vesicles and then in numerous newly created vesicles that accumulate in the cytoplasm and around the nucleus. Colony-forming assays showed EM and DnsEM to be equally cytotoxic to cultured DU 145 cells. Cellular uptake and subsequent manifestation of cytotoxicity are presumably dependent upon these vesicles. However, after uptake of DnsEM, its diffusion into the cytoplasm was observed.

Walker 256 rat mammary carcinoma cells resistant to chlorambucil (WR) exhibited an approximate 4-fold increase in glutathione S-transferase (GST) activity using 1-chloro-2,4-dinitrobenzene as compared to the sensitive parent cell line (WS). WR cells maintained without biannual exposure to chlorambucil (WRr) reverted to the sensitive phenotype and possessed GST levels equivalent to WS. Mitochondria, microsomes and cytosol were isolated from WS, WR and WRr cell lines and analyzed for their GST composition. GST activity in each subcellular compartment of resistant cells was increased over the sensitive cells. Antibodies raised against total rat liver cytosolic GST crossreacted in resistant cells with two microsomal proteins (25.7 kD and 29 kD). The 29 kD protein was not detected in microsomal fractions from either WS or WRr and this protein was found to be dissimilar from cytosolic GST subunits in its isoelectric point (pI 6.7) and migration on two-dimensional polyacrylamide gels. In addition, the 29 kD microsome-associated GST from WR cells was immunologically distinct from a 14 kD GST subunit previously identified in rat liver microsomes. These data implicate the induction of a specific microsomal GST subunit in WR cells following drug selection and suggest its potential involvement in the establishment of cellular resistance to chlorambucil.

We have investigated the ability of estramustine to bind to rat brain microtubule-associated proteins (MAPs) and purified MAP-2 in vitro. [3H]estramustine's relative affinity for tubulin and MAPs was assessed by gel filtration chromatography, immunoprecipitation and binding assays. Scatchard analysis demonstrated a specific affinity of the drug for MAP-2. Calculations from kinetic parameters and non-linear regression analysis gave a Kd of 15 microM, and a Bmax of 3.4 x 10(-7)M ml-1. Extrapolation of this value suggested that each MAP-2 molecule binds approximately 20 molecules of estramustine. Microtubule assembly studies and SDS-polyacrylamide gel electrophoresis revealed that at 20-60 microM levels, estramustine inhibited the association of MAPs with taxol microtubules. Turbidity (A350) studies further demonstrated that 20-60 microM-estramustine inhibited MAP-2-driven tubulin assembly and produced microtubule disassembly. Electron-microscopic studies confirmed the centrifugation and turbidity results. The data demonstrated that estramustine can bind MAPs and MAP-2 specifically, thereby inhibiting microtubule assembly.

A novel nitrosourea, 1-(2-chloroethyl)-3-[2-(dimethylaminosulfonyl)ethyl]-1-nitrosourea (TCNU) has been investigated with respect to cytotoxic mechanisms in rat and human cell lines which either possess (Mer+) or lack (Mer-) 0(6)-alkylguanine transferase activity. TCNU produced significantly greater cytotoxicity in the Mer- cells (Walker 256 rat breast carcinoma resistant to nitrogen mustards; human lung carcinoma A427) than in the Mer+ cells (Walker 256 wild-type; human lung carcinoma A594). This correlated with results generated by alkaline elution studies which showed that TCNU caused DNA interstrand crosslinks in A427 but not in A549 cells. Inhibition of glutathione reductase activity by TCNU demonstrated that in carbamoylating activity the drug was intermediate between chlorozotocin and 1,(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in both A427 and A549 cells. These data suggest that the presence of taurine in the drug structure does little to alter the cytotoxicity or the alkylating or carbamoylating properties of TCNU, and that any clinical advantages with TCNU will be the consequence of other factors.

Using polyclonal antibodies raised against a rat liver nuclear envelope protein, lamin protein A, the nuclear matrix proteins of a Walker 256 rat mammary carcinoma wild-type (WS) and a selected cell line with acquired resistance to nitrogen mustards (WR) were found to possess antigenic determinants which were recognized by the antibodies. In one-dimensional immunoblotting analysis, the nuclear matrix protein fractions of both cell lines revealed a common band at Mr 75,000; however only the WS nuclear matrix protein fraction contained a broad band at approximately Mr 70,000. Two-dimensional gel blotting studies of these proteins showed that this Mr 70,000 WS protein had a pI of approximately 7.5. Immunoprecipitation analysis revealed that the altered mobility of this protein could be a function of phosphorylation. The nuclear matrix proteins from both WS and WR cells were shown to bind 3':5'-cyclic adenylic acid (cAMP), as judged by photoaffinity labeling and gel electrophoresis studies. The WS nuclear matrix proteins showed a quantitatively greater level of cAMP binding compared to WR, with predominant binding to proteins with molecular weights of 45,000, 55,000, and 70,000. In WR cells, there was no cAMP binding in the Mr 70,000 region. These data indicate that the Mr 70,000 nuclear matrix lamin proteins are antigenically similar in WS and WR but differ in that the WR protein is hypophosphorylated and does not bind cAMP.

Over two decades, experience with estramustine has provided limited data which support an estrogenic mechanism of action and no data which indicate the nitrogen mustard involvement in the cytotoxic properties of the drug. Consideration of the carbamate-ester portion of estramustine supports the pharmacokinetic evidence that estramustine has a long half life since enzymatic hydrolysis of the carbamate is an uncommon event. Using a variety of immunocytochemical and cellular morphology procedures, estramustine per se has been found to express anti-cytoskeletal properties through non-covalent binding to microtubule associated proteins (MAP's). In both fish erythrophores and in dividing human prostatic carcinoma cells, estramustine exerts an antimicrotubule effect at micromolar concentrations. Thus, estramustine possesses unique pharmacology and protein binding specificity. As such, it should not be classified as an alkylating agent. The estrogenic effects, while possibly of relevance to clinical administration, are not the primary mechanism by which the drug exerts cytotoxicity.

We have examined the pharmacological characteristics of three dinitrosated isomers of N,N'-bis[N(2-chloroethyl)-N-carbamoyl]cystamine [CNCC-(D), 1C1G1325] differing in the relative positions of the nitroso substituents [CNCC-(C), (1,1' dinitroso); CNCC-(S), 3,3' dinitroso); and CNCC-(M), (1,3'-dinitroso)] and which were designed to be subject to preferential bioreductive activation in hypoxic tumors. The decomposition products of the isomers formed under physiological conditions [both in the absence and in the presence of dithiothreitol (DDT)] were identified and quantified. For example, CNCC-(S) in phosphate buffer, pH 7.0, and 37 degrees gave rise to 2-chloroethylisocyanate, bis(2-chloroethyl)urea and bis(2-hydroxyethyl)disulfide, whereas in the presence of DTT it afforded 2-chloroethylisocyanate, bis(2-chloroethyl)urea, bis(2-hydroxyethyl) disulfide, thiirane and 2-mercaptoethanol. Control aqueous decomposition profiles were performed with two known metabolites of CNCC, namely 3-(2-chloroethyl)-1-(2-thioethyl)-1-nitrosourea and 3-(2-chloroethyl)-1-(2-methylthioethyl)-1-nitrosourea. CNCC-(C) caused 20% interstrand cross-linking of lambda-DNA in 2 hr, whereas in the presence of DTT the extent of cross-linking increased to 38% in the same time period. In contrast, isomer (S) showed no detectable cross-linking in 7 hr. This thiol potentiation of cross-linking which is observed with other 2-chloroethylnitrosoureas is explained by nucleophilic attack at the carbonyl group and subsequent stereoelectronically controlled decomposition of the tetrahedral intermediate. The relative extents of carbamoylating activity of the CNCC isomers were obtained using a [14C]-lysine assay which showed (S) approximately equal to (M) greater than (C). Inhibition of glutathione reductase for both Walker 256 resistant (WR) and Walker 256 sensitive (WS) strains showed that isomer (S) inactivated the enzyme more effectively than isomer (C) in accord with the carbamoylating activity results. The higher carbamoylators (S) and (M) also showed greater effects on the intracellular thiol pools in both WR and WS cells indicative of sulfhydryl conjugation and efflux and/or inhibition of the GSH metabolic enzymes. In vitro cytotoxicity studies with human DU 145 prostatic carcinoma cells showed the isomer cytotoxicity was (M) greater than (C) greater than (S) over a 24-hr incubation period. The reduced cytotoxic potential of CNCC-(S) in both the Walker 256 cells and in the human prostatic carcinoma cells may be a function of an interaction between GSH and the drug thereby protecting other more critical nucleophilic targets within the nucleus.

Walker 256 rat carcinosarcoma cell lines sensitive (WS) or resistant (WR) to bifunctional nitrogen mustards have modal chromosome numbers of 60 and 55 respectively. Karyotype analysis revealed that these cell lines have retained the major marker chromosomes present in the original in vivo Walker tumours. One new marker chromosome, a metacentric, was found in the WR cell line. C-banding revealed that in the WS cell line the secondary constriction of the marker chromosome was stained, whereas no staining was found on this chromosome in the WR cell line. Three autosomes containing very prominent non-centromeric C-bands were present in WS but not in the WR cell line which has 2 other chromosomes with minor C-bands. As non-centromeric C-bands do not occur in the normal rat karyotype, these are easily identifiable specific tumour markers for these two cell lines.