Increased sensitivity to platinum drugs of cancer cells with acquired resistance to trabectedin.

BACKGROUND: In order to investigate the mechanisms of acquired resistance to trabectedin, trabectedin-resistant human myxoid liposarcoma (402-91/T) and ovarian carcinoma (A2780/T) cell lines were derived and characterised in vitro and in vivo. METHODS: Resistant cell lines were obtained by repeated exposures to trabectedin. Characterisation was performed by evaluating drug sensitivity, cell cycle perturbations, DNA damage and DNA repair protein expression. In vivo experiments were performed on A2780 and A2780/T xenografts. RESULTS: 402-91/T and A2780/T cells were six-fold resistant to trabectedin compared with parental cells. Resistant cells were found to be hypersensitive to UV light and did not express specific proteins involved in the nucleotide excision repair (NER) pathway: XPF and ERCC1 in 402-91/T and XPG in A2780/T. NER deficiency in trabectedin-resistant cells was associated with the absence of a G2/M arrest induced by trabectedin and with enhanced sensitivity (two-fold) to platinum drugs. In A2780/T, this collateral sensitivity, confirmed in vivo, was associated with an increased formation of DNA interstrand crosslinks. CONCLUSIONS: Our finding that resistance to trabectedin is associated with the loss of NER function, with a consequent increased sensitivity to platinum drugs, provides the rational for sequential use of these drugs in patients who have acquired resistance to trabectedin.

Lack of unique neuropathology in amyotrophic lateral sclerosis associated with p.K54E angiogenin (ANG) mutation.

AIMS: Five to 10% of cases of amyotrophic lateral sclerosis are familial, with the most common genetic causes being mutations in the C9ORF72, SOD1, TARDBP and FUS genes. Mutations in the angiogenin gene, ANG, have been identified in both familial and sporadic patients in several populations within Europe and North America. The aim of this study was to establish the incidence of ANG mutations in a large cohort of 517 patients from Northern England and establish the neuropathology associated with these cases. METHODS: The single exon ANG gene was amplified, sequenced and analysed for mutations. Pathological examination of brain, spinal cord and skeletal muscle included conventional histology and immunohistochemistry. RESULTS: Mutation screening identified a single sporadic amyotrophic lateral sclerosis case with a p.K54E mutation, which is absent from 278 neurologically normal control samples. The clinical presentation was of limb onset amyotrophic lateral sclerosis, with rapid disease progression and no evidence of cognitive impairment. Neuropathological examination established the presence of characteristic ubiquitinated and TDP-43-positive neuronal and glial inclusions, but no abnormality in the distribution of angiogenin protein. DISCUSSION: There is only one previous report describing the neuropathology in a single case with a p.K17I ANG mutation which highlighted the presence of eosinophilic neuronal intranuclear inclusions in the hippocampus. The absence of this feature in the present case indicates that patients with ANG mutations do not always have pathological changes distinguishable from those of sporadic amyotrophic lateral sclerosis.

Osimertinib and anti-HER3 combination therapy engages immune dependent tumor toxicity via STING activation in trans.

Over the past decade, immunotherapy delivered novel treatments for many cancer types. However, lung cancer still leads cancer mortality, and non-small-cell lung carcinoma patients with mutant EGFR cannot benefit from checkpoint inhibitors due to toxicity, relying only on palliative chemotherapy and the third-generation tyrosine kinase inhibitor (TKI) osimertinib. This new drug extends lifespan by 9-months vs. second-generation TKIs, but unfortunately, cancers relapse due to resistance mechanisms and the lack of antitumor immune responses. Here we explored the combination of osimertinib with anti-HER3 monoclonal antibodies and observed that the immune system contributed to eliminate tumor cells in mice and co-culture experiments using bone marrow-derived macrophages and human PBMCs. Osimertinib led to apoptosis of tumors but simultaneously, it triggered inositol-requiring-enzyme (IRE1α)-dependent HER3 upregulation, increased macrophage infiltration, and activated cGAS in cancer cells to produce cGAMP (detected by a lentivirally transduced STING activity biosensor), transactivating STING in macrophages. We sought to target osimertinib-induced HER3 upregulation with monoclonal antibodies, which engaged Fc receptor-dependent tumor elimination by macrophages, and STING agonists enhanced macrophage-mediated tumor elimination further. Thus, by engaging a tumor non-autonomous mechanism involving cGAS-STING and innate immunity, the combination of osimertinib and anti-HER3 antibodies could improve the limited therapeutic and stratification options for advanced stage lung cancer patients with mutant EGFR.

Defective repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cell tumours.

Metastatic cancer in adults usually has a fatal outcome. In contrast, advanced testicular germ cell tumours are cured in over 80% of patients using cisplatin-based combination chemotherapy [1]. An understanding of why these cells are sensitive to chemotherapeutic drugs is likely to have implications for the treatment of other types of cancer. Earlier measurements indicate that testis tumour cells are hypersensitive to cisplatin and have a low capacity to remove cisplatin-induced DNA damage from the genome [2] [3]. We have investigated the nucleotide excision repair (NER) capacity of extracts from the well-defined 833K and GCT27 human testis tumour cell lines. Both had a reduced ability to carry out the incision steps of NER in comparison with extracts from known repair-proficient cells. Immunoblotting revealed that the testis tumour cells had normal amounts of most NER proteins, but low levels of the xeroderma pigmentosum group A protein (XPA) and the ERCC1-XPF endonuclease complex. Addition of XPA specifically conferred full NER capacity on the testis tumour extracts. These results show that a low XPA level in the testis tumour cell lines is sufficient to explain their poor ability to remove cisplatin adducts from DNA and might be a major reason for the high cisplatin sensitivity of testis tumours. Targeted inhibition of XPA could sensitise other types of cells and tumours to cisplatin and broaden the usefulness of this chemotherapeutic agent.

Repair of intermediate structures produced at DNA interstrand cross-links in Saccharomyces cerevisiae.

Bifunctional alkylating agents and other drugs which produce DNA interstrand cross-links (ICLs) are among the most effective antitumor agents in clinical use. In contrast to agents which produce bulky adducts on only one strand of the DNA, the cellular mechanisms which act to eliminate DNA ICLs are still poorly understood, although nucleotide excision repair is known to play a crucial role in an early repair step. Using haploid Saccharomyces cerevisiae strains disrupted for genes central to the recombination, nonhomologous end-joining (NHEJ), and mutagenesis pathways, all these activities were found to be involved in the repair of nitrogen mustard (mechlorethamine)- and cisplatin-induced DNA ICLs, but the particular pathway employed is cell cycle dependent. Examination of whole chromosomes from treated cells using contour-clamped homogenous electric field electrophoresis revealed the intermediate in the repair of ICLs in dividing cells, which are mostly in S phase, to be double-strand breaks (DSBs). The origin of these breaks is not clear since they were still efficiently induced in nucleotide excision and base excision repair-deficient, mismatch repair-defective, rad27 and mre11 disruptant strains. In replicating cells, RAD52-dependent recombination and NHEJ both act to repair the DSBs. In contrast, few DSBs were observed in quiescent cells, and recombination therefore seems dispensable for repair. The activity of the Rev3 protein (DNA polymerase zeta) is apparently more important for the processing of intermediates in stationary-phase cells, since rev3 disruptants were more sensitive in this phase than in the exponential growth phase.

Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells.

The mechanisms by which DNA interstrand cross-links (ICLs) are repaired in mammalian cells are unclear. Studies in bacteria and yeasts indicate that both nucleotide excision repair (NER) and recombination are required for their removal and that double-strand breaks are produced as repair intermediates in yeast cells. The role of NER and recombination in the repair of ICLs induced by nitrogen mustard (HN2) was investigated using Chinese hamster ovary mutant cell lines. XPF and ERCC1 mutants (defective in genes required for NER and some types of recombination) and XRCC2 and XRCC3 mutants (defective in RAD51-related homologous recombination genes) were highly sensitive to HN2. Cell lines defective in other genes involved in NER (XPB, XPD, and XPG), together with a mutant defective in nonhomologous end joining (XRCC5), showed only mild sensitivity. In agreement with their extreme sensitivity, the XPF and ERCC1 mutants were defective in the incision or "unhooking" step of ICL repair. In contrast, the other mutants defective in NER activities, the XRCC2 and XRCC3 mutants, and the XRCC5 mutant all showed normal unhooking kinetics. Using pulsed-field gel electrophoresis, DNA double-strand breaks (DSBs) were found to be induced following nitrogen mustard treatment. DSB induction and repair were normal in all the NER mutants, including XPF and ERCC1. The XRCC2, XRCC3, and XRCC5 mutants also showed normal induction kinetics. The XRCC2 and XRCC3 homologous recombination mutants were, however, severely impaired in the repair of DSBs. These results define a role for XPF and ERCC1 in the excision of ICLs, but not in the recombinational components of cross-link repair. In addition, homologous recombination but not nonhomologous end joining appears to play an important role in the repair of DSBs resulting from nitrogen mustard treatment.

Excision repair of nitrogen mustard-DNA adducts in Saccharomyces cerevisiae.

The bifunctional alkylating anticancer drug nitrogen mustard forms a variety of DNA lesions, including monoadducts and intrastrand and interstrand crosslinks. Although it is known that nucleotide excision repair (NER) is important in processing these adducts, the role of the other principal excision repair pathway, base excision repair (BER) is less well defined. Using isogenic Saccharomyces cerevisiae strains disrupted for a variety of NER and BER genes we have examined the relative importance of the two pathways in the repair of nitrogen mustard adducts. As expected, NER defective cells (rad4 and rad14 strains) are extremely sensitive to the drug. One of the BER mutants, a 3-methyladenine glycosylase defective (mag1) strain also shows significant hypersensitivity. Using a rad4/mag1 double mutant it is shown that the two excision repair pathways are epistatic to each other for nitrogen mustard sensitivity. Furthermore, both rad14 and mag1 disruptants show elevated levels of nitrogen mustard-induced forward mutation. Measurements of repair rates of nitrogen mustard N-alkylpurine adducts in the highly transcribed RPB2 gene demonstrate defects in the processing of mono-adducts in rad4, rad14 and mag1 strains. However, there are differences in the kinetics of adduct removal in the NER mutants compared to the mag1 strain. In the mag1 strain significant repair occurs within 1 h with evidence of enhanced repair on the transcribed strand. Adducts however accumulate at later times in this strain. In contrast, in the NER mutants repair is only evident at times greater than 1 h. In a mag1/rad4 double mutant damage accumulates with no evidence of repair. Comparison of the rates of repair in this gene with those in a different genomic region indicate that the contributions of NER and BER to the repair of nitrogen mustard adducts may not be the same genome wide.

DNA damage and cytotoxicity of 2-chloroethyl (methylsulfonyl)methanesulfonate (NSC 338947) produced in human colon carcinoma cells with or without methylating agent pretreatment.

2-Chloroethyl (methylsulfonyl)methanesulfonate (ClEtSoSo) was more toxic to the BE (Mer-) cell line than to the HT-29 (Mer+) colon carcinoma. The sensitivity of the BE cells closely paralleled the induction of DNA interstrand cross-links by ClEtSoSo. No DNA interstrand crosslink formation was detected in the HT-29 cells after exposure to ClEtSoSo. Pretreatment of the HT-29 cells with methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine or streptozotocin increases their sensitivity to ClEtSoSo. Little or no increase in the toxicity of ClEtSoSo was found in BE cells after methylating agent pretreatment. Despite the increase in cell killing, no DNA interstrand cross-links were induced by ClEtSoSo after N-methyl-N'-nitro-N-nitrosoguanidine pretreatment. In contrast, streptozotocin pretreatment allowed ClEtSoSo to form DNA interstrand cross-links in HT-29 cells. The production of DNA strand breaks by ClEtSoSo was observed in HT-29 cells both with and without methylating agent pretreatment. These results suggest that the mechanism of ClEtSoSo may differ from other chloroethylating agents such as the chloroethylnitrosoureas. In addition, there may be a difference in the mechanism by which streptozotocin or N-methyl-N'-nitro-N-nitrosoguanidine pretreatment causes an increased cell killing in a previously resistant human colon carcinoma cell line.

DNA reactivity and in vitro cytotoxicity of the novel antitumor agent 1,5,2,4-dioxadithiepane-2,2,4,4-tetraoxide (NSC-348948) in human embryo cells.

1,5,2,4-Dioxadithiepane-2,2,4,4-tetraoxide (cyclic-SoSo) is structurally a novel synthetic compound but may functionally act as an alkylating agent. The effects of cyclic-SoSo on DNA were studied in IMR-90 and VA-13 human embryo cells by means of DNA alkaline elution analysis. In contrast to a number of bifunctional alkylating agents, cyclic-SoSo produced no DNA-DNA interstrand cross-links in either cell line, even at concentration which produced a greater than 3 log cell kill. At equimolar concentrations cyclic-SoSo induced DNA-protein cross-links in both cell lines to a similar extent. Frank DNA breaks and alkali-labile DNA lesions were detected in both cell lines. A greater quantity of strand breaks appeared in the IMR-90 than in the VA-13 cell line after exposure to cyclic-SoSo. However, cyclic-SoSo was more cytotoxic to the VA-13 cell line in vitro than to the IMR-90 cell line. Thus cyclic-SoSo may not be a typical bifunctional alkylating agent in that its mechanism of action does not appear to involve DNA interstrand cross-linking.

Combined effects of streptozotocin and mitozolomide against four human cell lines of the Mer+ phenotype.

The majority of human tumor cell lines are proficient in the repair of guanine O6-alkylations (designated Mer+) and are thus capable of preventing the cytotoxic effects of chloroethylating agents. It has been proposed that in these cells guanine O6-chloroethylations are rapidly removed by the enzyme O6-alkylguanine DNA alkyltransferase before the formation of DNA interstrand cross-links can occur. In this study pretreatment of four Met+ human cells (A2182 lung carcinoma, A375 melanoma, HT-29 colon carcinoma, and IMR-90 normal lung fibroblasts) with the DNA methylating agent streptozotocin apparently saturates the monoadduct repair system and allows mitozolomide to form interstrand cross-links in these cells. The inhibition of the alkyltransferase results in the continued presence of guanine O6-chloroethylations which then undergo a series of reactions that lead to DNA interstrand cross-link formation. As observed by colony forming assays, streptozotocin pretreatment causes a dramatic increase in the sensitivity of these four Mer+ cell lines to the cytotoxic effects of mitozolomide. These results indicate that a combination of streptozotocin pretreatment followed by mitozolomide may be useful in the treatment of human cancer.

2-Chloroethyl (methylsulfonyl)methanesulfonate (NSC-338947), a more selective DNA alkylating agent than the chloroethylnitrosoureas.

The novel chloroethylating agent 2-chloroethyl (methylsulfonyl)methanesulfonate (CIEtSoSo) has been shown to act like the chloroethylnitrosoureas (CIEtNu's) in its DNA damaging and cytotoxic effects in human cell lines and has similar activity to the CIEtNu's in the National Cancer Institute antitumor screening tests. Its simpler chemistry, however, suggests that it may alkylate DNA more selectively than do the CIEtNu's. The DNA base adducts produced in calf thymus DNA by CIEtSoSo have been compared to a representative, non-carbamoylating CIEtNu, 1-(2-chloroethyl)-3-(cis-2-OH)cyclohexyl-1-nitrosourea, using high-pressure liquid chromatography. Two major modified base peaks were observed from the nitrosourea treated sample which have been subsequently identified by high-pressure liquid chromatography comparison of synthesized standards and by electron impact gas chromatography/mass spectrometric analysis to be 7-hydroxyethylguanine and 7-chloroethylguanine. In contrast only 7-chloroethylguanine was obtained from the CIEtSoSo treated DNA at equimolar doses. Thus CIEtSoSo was found to be more specific in its reaction with DNA in that it produced less variety of products than the nitrosourea, with no apparent generation of hydroxyethyl products, which are major side reactions of the CIEtNu's.

DNA sequence selectivity of guanine-N7 alkylation by three antitumor chloroethylating agents.

The DNA sequence selectivities of guanine-N7 alkylation produced by three chloroethylating antitumor agents, 1-(2-chloroethyl)-3-(cis-2-hydroxy) cyclohexyl-1-nitrosourea (cis-2-OH CCNU), 2-chloroethyl (methylsulfonyl)methanesulfonate, and 8-carbamoyl-3-(2-chloroethyl)imidazo-[5,1-d]-1,2,3,5-tetrazin-4(3H )-one (mitozolomide), were examined using a modification of the Maxam and Gilbert sequencing technique. In a region of pBR322 DNA, 2-chloroethyl (methylsulfonyl)methanesulfonate produced approximately the same degree of alkylation at all guanines. cis-2-OH CCNU, however, preferentially alkylated the middle guanines in runs of three or more guanines; the intensity of the reaction increased with the number of adjacent guanines in the DNA sequence. Mitozolomide produced the same pattern of preferential alkylation but not as intensely as cis-2-OH CCNU. Three other nitrosoureas, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, 1-(2-fluorethyl)-3-cyclohexyl-1-nitrosourea, and 1-(2-chloroethyl)-1-nitrosourea gave similar patterns of alkylation to that of cis-2-OH CCNU at pH 7.2. The ratio of 7-hydroxyethylguanine to 7-chloroethylguanine was approximately the same following treatment of the synthetic polymers dGn X dCn and (dG X dC)n with cis-2-OH CCNU, indicating that 7-chloroethylation and 7-hydroxyethylation were enhanced similarly by the presence of adjacent guanines. Ethylnitrosourea produced relatively little alkylation preference. The results suggest that the alkylating intermediates, 2-chloroethyldiazohydroxide and 2-hydroxyethyldiazohydroxide, tend to react preferentially with those guanine-N7 positions the electronegativity of which is enhanced by the presence of neighboring guanines. This is consistent with the presence of cationic character in the alkylating centers of these intermediates. 2-Chloroethyl (methylsulfonyl)methanesulfonate and ethyldiazohydroxide would not be expected to have strong cationic character, in agreement with their lack of sequence selectivity.

Association of DNA strand breaks with accelerated terminal differentiation in mouse epidermal cells exposed to tumor promoters.

We have studied the appearance of single strand breaks (SSB) in DNA of mouse keratinocytes exposed in vitro to various tumor promoters. Mouse basal keratinocytes were selectively cultured in low calcium medium, prelabeled with [14C]thymidine, exposed to test agents, and SSB quantified by alkaline elution. 12-O-Tetradecanoylphorbol-13-acetate (TPA) caused a dose-dependent (10(-9)-10(-7) M) increase in SSB after 24 h but not after shorter exposures. DNA containing TPA-induced SSB was found only in cells which had detached from the culture plate as a consequence of TPA-induced terminal differentiation. Attached cells, resistant to the differentiation-inducing effects of TPA, had the low level of SSB found in DNA from vehicle-treated control cells. Attached cells were resistant to the formation of SSB and to induced differentiation when reexposed to TPA. Other tumor-promoting phorbol esters, mezerein and retinyl phorbol acetate, also produced SSB in detached cells, whereas phorbol or resiniferatoxin caused neither SSB or cell detachment. Retinoic acid, which blocks the induction of differentiation by TPA, inhibited the production of SSB by TPA; however, fluocinolone acetonide, chymostatin, catalase, or superoxide dismutase blocked neither TPA-induced SSB nor terminal differentiation. Epidermal cell lines resistant to TPA-induced differentiation were also resistant to SSB production by TPA. Benzoyl peroxide (BP) (10(-4) M) induced SSB in basal keratinocytes within 1 h, and attached cells showed extensive SSB by 12 h. Retinoic acid had only a slight effect on BP-induced SSB, and 1 of 3 TPA-resistant cell lines developed SSB when exposed to BP. These results suggest that TPA-induced SSB in epidermal cells are an indirect consequence of the induction of terminal differentiation, whereas BP produces SSB by a more direct mechanism of DNA damage.

Differential cytotoxicity and DNA-damaging effects produced in human cells of the Mer+ and Mer- phenotypes by a series of 1-aryl-3-alkyltriazenes.

A series of arylalkyltriazenes has been investigated for its differential cytotoxicity towards the HT-29 (Mer+) and BE (Mer-) cell lines and for its ability to cause DNA strand breaks and cross-links. A monomethyltriazene (MMPT) and some hydroxymethyltriazene derivatives capable of generating the monomethyltriazene in situ were preferentially cytotoxic to the BE cell line when compared with the HT-29 cell line. The differential toxicity of MMPT is very similar to the analogous monochloroethyltriazene. In contrast, the dimethyl- and monoethyltriazenes in the series display reduced toxicity towards the BE cell line with little or no differential toxicity between the BE and HT-29 cell lines. MMPT and monochloroethyltriazene caused single strand DNA breaks in the two cell lines, whereas little or no DNA strand breaks were observed in either cell line after exposure to the monoethyl- or dimethyltriazene. However, these lesions could not account for the differential cytotoxicity observed. In measurements of DNA interstrand cross-linking none of the agents tested, including monochloroethyltriazene and MMPT, was found to cause such linkages. In contrast to previous results obtained with bifunctional monochloroethylating agents, which produced a similar differential cytotoxicity between these two cell lines, our results tend to suggest that lesions other than DNA interstrand cross-links may be responsible for the mechanisms of cell killing by chloroethylating agents.

Photosensitization of human leukemic cells by anthracenedione antitumor agents.

1,4-Diamino-substituted anthraquinone antitumor agents (mitoxantrone and ametantrone) and structurally related 1,5- and 1,8-diamino-substituted compounds (AM1 and AM2) were tested for their ability to photosensitize human leukemic cells in culture. Viability was measured using the 3,4,5-dimethylthiazol-2,5-diphenyl tetrazolium bromide assay, and DNA and membrane damage were assessed. Following a 1-h exposure to AM2, a dose of drug required to give 50% loss of cell viability (53 microM) was obtained in the dark, which was reduced to approximately 2.4 microM following illumination for 2 min (lambda greater than 475 nm), a dose of light that was completely nontoxic to the cells in the absence of drug. A shift in the cell viability curve was also observed for AM1 but, under identical conditions, the dose modification was only 8.9. In contrast, neither ametantrone nor mitoxantrone gave a decreased viability upon illumination. DNA single-strand breaks as measured by alkaline elution correlated with cell viability. Frank DNA single-strand breaks were produced by AM2 and light, suggesting the production of free radicals. The strand breaks produced by AM2 in the dark and by mitoxantrone (with or without illumination) were protein concealed. No evidence of photo-induced membrane damage, as determined by transport of the model amino acid cycloleucine, could be observed even at supralethal doses.

Liblomycin-mediated DNA cleavage in human head and neck squamous carcinoma cells and purified DNA.

Liblomycin (LBM), a novel bleomycin analogue, and bleomycin A2 (BLM A2) were compared with respect to their relative potential to inhibit growth in a human head and neck squamous carcinoma cell line and to produce DNA damage within cellular DNA and nuclei DNA and against isolated naked DNA. Against the BLM-sensitive cell line 183A, the concentration of LBM that inhibits cell growth by 50% was 1.1 microM for a 30-min drug exposure, while it was 23 microM for BLM A2. Drug-mediated DNA double-strand cleavage within cells was compared with the relative ability of these drugs to produce DNA cleavage in isolated 183A cell nuclei. Though 30-min exposures of cells to equimolar concentrations of both drugs resulted in 4-fold greater cellular DNA damage by LBM than BLM A2, the two drugs were nearly equipotent in producing DNA injury within isolated nuclei. Against Simian virus 40 DNA, however, LBM was 10-fold less effective than BLM A2 in producing Forms II and III DNA from Form I DNA. Radioactivity from either [3H]BLM A2 or 125I-LBM found associated with cells after a 30-min incubation period was also assessed in the 183A cell line. The exposure of cells to radiolabeled drug (1 microM) resulted in a 71-fold greater amount of cell-associated radioactivity for LBM than for BLM A2. The relative abilities of the 183A cell line to partially reseal LBM- or BLM A2-mediated DNA double-strand breaks were also assessed. No preferential repair of overall drug-mediated DNA injury, however, was observed. Finally, drug-mediated specific cleavage sites on pBR322 DNA were determined. At doses that gave the same extent of DNA cleavage, both BLM A2 and LBM gave similar patterns of strand scission, although minor differences were observed. Taken together, these data demonstrate that the greater efficacy of LBM against the BLM-sensitive head and neck squamous cell line is due mainly to LBM's greater association with cells over a defined time period, even though the DNA cleaving ability of LBM is relatively lower than that of BLM A2.

Targeting of tumor cells and DNA by a chlorambucil-spermidine conjugate.

Many tumor cells, including murine ADJ/PC6 plasmacytoma cells, possess an active energy dependent polyamine uptake system which selectively accumulates endogenous polyamines and structurally related compounds. We have attempted to target the cytotoxic drug chlorambucil to a tumor possessing this uptake system by conjugating it to the polyamine spermidine. Furthermore, since polyamines have a high affinity for DNA, the attachment of spermidine to chlorambucil should also facilitate its targeting to DNA. This was supported by the observation that the chlorambucil-spermidine conjugate was approximately 10,000-fold more active than chlorambucil at forming interstrand crosslinks with naked DNA. In vitro cytotoxicity and in vivo antitumor studies were carried out using the ADJ/PC6 plasmacytoma. In vitro, using [3H]thymidine incorporation to assess cell viability following a 1-h exposure to control and polyamine depleted ADJ/PC6 cells, chlorambucil-spermidine was 35- and 225-fold, respectively, more toxic than chlorambucil. The increased toxicity of the conjugate compared to chlorambucil was possibly due to enhanced DNA binding and/or facilitated uptake via the polyamine uptake system. The enhanced toxicity of the conjugate but not chlorambucil by prior polyamine depletion with difluoromethylornithine, together with the observation that the conjugate but not chlorambucil competitively inhibited spermidine uptake into tumor cells, supported the suggestion that the conjugate utilized the polyamine uptake system. In vivo following a single i.p. dose, the conjugate was 4-fold more potent than chlorambucil in its ability to inhibit ADJ/PC6 tumor growth in BALB/c mice. However, the therapeutic index was not increased. Our results support the hypothesis that polyamines linked to cytotoxics facilitate their entry into tumor cells possessing a polyamine uptake system and increase their selectivity to DNA. This may have therapeutic application in the delivery of cytotoxic agents linked to polyamines to certain tumors.

Relationship between topoisomerase II level and chemosensitivity in human tumor cell lines.

Patients with metastatic testis tumors are generally curable using chemotherapy, whereas those with disseminated bladder carcinomas are not. We have compared levels of the nuclear enzyme topoisomerase II in three testis (SuSa, 833K, and GH) and three bladder (RT4, RT112, and HT1376) cancer cell lines which differ in their sensitivity to chemotherapeutic agents. The testis cell lines were more sensitive than the bladder lines to three drugs whose cytotoxicity is mediated in part by inhibiting topoisomerase II: amsacrine; Adriamycin; and etoposide (VP16). The frequency of DNA strand breaks induced by amsacrine was higher (1.5- to 13-fold) in the testis cells than in the bladder cells. The level of topoisomerase II-mediated DNA strand breakage in vitro, measured by filter trapping of amsacrine-induced protein:DNA cross-links, was similarly higher in nuclear extracts from the testis than the bladder cells. Western blot analysis showed a generally higher level of topoisomerase II protein in testis than in bladder cell nuclear extracts. Topoisomerase II protein expression broadly correlated with drug-induced strand breakage in both protein extracts and whole cells, but not with population doubling time. However, despite a 2- to 20-fold increased sensitivity to the different topoisomerase II inhibitors, the testis line 833K had a less than 2-fold higher level of topoisomerase II protein than that of the bladder line RT4. These results indicate that the level of expression of topoisomerase II is an important determinant of the relative chemosensitivity of testis and bladder tumor cell lines, but that additional factors must contribute to the extreme chemosensitivity of testis cells.

bcl-2 modulation of apoptosis induced by anticancer drugs: resistance to thymidylate stress is independent of classical resistance pathways.

The hypothesis was tested that expression of bcl-2 could provide protection against apoptosis induced by cytotoxic drugs via a mechanism which was different from the classical determinants of drug resistance. Sensitivity and resistance to inhibitors of thymidylate synthase (EC 2.1.1. 45) were chosen for study since these drugs have a well-defined and quantifiable locus of action with similarly well defined biochemical sequelae resulting from enzyme inhibition. Human lymphoma cells transfected with the vector alone readily underwent apoptosis after a 36-h exposure to various drugs. For example, 5-fluorodeoxyuridine (0.1 microM) induced 67% apoptosis in vector control cells 24 h after removal of the drug. In contrast, cells treated under identical conditions, but which expressed the bcl-2 protein, showed only basal levels of apoptosis (8%), with no significant fall in viability. Similar results were obtained using two quinazoline-based inhibitors of thymidylate synthase, N10-propargyl-5,8-dideazafolic acid (CB3717) and ICI M247496. Determinants of resistance to these three drugs were investigated. Analysis of the cell cycle, thymidylate synthase levels, and activity showed these to be unchanged by expression of bcl-2. Addition of the drugs brought about equivalent inhibition of proliferation in the presence or absence of bcl-2 expression. 5-Fluorodeoxyuridine treatment reduced TTP synthesis, induced strand breaks in nascent DNA, measured by alkaline elution, and increased the synthesis of thymidylate synthase; these changes preceded the onset of apoptosis and were identical in the vector controls and bcl-2 transfectants. Resistance to thymidylate stress in bcl-2-expressing cells therefore occurred by a mechanism different from those which classically define resistance to this type of cytotoxic drug.

Mechanism of DNA strand breakage by piperidine at sites of N7-alkylguanines.

The volatile, secondary amine piperidine is used in the Maxam-Gilbert chemical method of DNA sequencing to create strand breaks in DNA at sites of damaged bases. As such it is often used in generalized studies of DNA damage to identify 'alkali-labile lesions'. We confirm the mechanism proposed by Maxam and Gilbert (Maxam, A. and Gilbert, W. (1980) Methods Enzymol. 65, 499-560) by which aqueous piperidine creates strand breaks at sites of N7-guanine alkylations: alkaline conditions catalyze rupture of the C8-N9 bond, forming a formamido-pyrimidine structure which is displaced from the ribose moiety by piperidine. In keeping with this mechanism, the tertiary amine, N-methylpiperidine, does not catalyze the formation of strand breaks in alkylated DNA. Our data confirm the prediction that high pH in and of itself will not create strand breaks at sites of N7-alkylguanines.