Nitrogen Mustard Treatment Research Articles

Choline Kinase a Is Required for Alkylating Agent Resistance in T-Cell Acute Lymphoblastic Leukemia

Conventional chemotherapy is responsible for most clinical cures achieved in oncology, but these drugs are toxic, and resistance is a major problem. Indeed,10-20% of children and >50% of adults with T-cell acute lymphoblastic leukemia (T-ALL) exhibit resistance to front-line therapy, leading to a dismal prognosis. Improved treatment options are needed for these patients. To identify mechanisms of alkylating agent resistance, we performed a genome-wide CRISPR screen in T-ALL cells treated with the alkylating agent nitrogen mustard, the active functional group of drugs such as cyclophosphamide and melphalan. The results revealed a number of internal positive controls, including genes implicated in the Fanconi and other DNA repair pathways. This screen also implicated a role for choline kinase A (CHKA) in nitrogen mustard resistance. CHKA is best-known for its ability to catalyze phosphocholine synthesis, a key step in phosphatidylcholine biosynthesis, and has no known role in DNA damage repair. We found that CHKA shRNA knockdown induces profound sensitivity to nitrogen mustard treatment in leukemia cells (p= 0.0012), but not in normal hematopoietic progenitor CD34+ cells. This effect was rescued by restoring expression of wild-type CHKA, but not by an ATPase-deficient CHKA point mutant. This indicates that CHKA kinase activity is required for resistance. We next assessed the role of CHKA in resistance to a panel of therapeutics with distinct mechanisms of action. We found that CHKA loss induced selective sensitivity to drugs that induce bulky DNA adducts. By contrast, CHKA had little effect on drugs that induce other forms of DNA damage, such as free radical damage or double-strand DNA breaks. Studies to elucidate the mechanism through which CHKA mediates resistance to these drugs are underway. To investigate the therapeutic potential of CHKA inhibition, we tested a small molecule CHKA inhibitor (CK2) in combination with alkylating agents in a panel of patient-derived T-ALL models. In vitro, we found that the pharmacologic inhibition of CHKA is synergistic with alkylating agent treatment with a bliss score of 16.683 for PDX9 and 21.982 for PDX CBAT (score >10 indicating strong synergy). We then tested the potential of this approach in these T-ALL PDX models when engrafted in vivo in NRG mice and treated with CK2 and cyclophosphamide alone or in combination. We found that the addition of CK2 further improved the survival benefit of cyclophosphamide in this model (p= 0.0260). Taken together, our findings support a new role of choline kinase A in alkylating agent resistance in T-ALL and provide a rationale to explore the possibility of increasing the efficacy of conventional chemotherapy in the context of drug-resistant leukemias by combining CHKA inhibition with alkylating agents.

Secondary cancers, comorbidities and mortality associated with nitrogen mustard therapy in patients with mycosis fungoides: a 30-year population-based cohort study

Topical nitrogen mustard is a widely used therapy in patients with mycosis fungoides (MF). However, it remains controversial whether nitrogen mustard therapy is associated with increased risk of secondary cancers and chronic pulmonary diseases in patients with MF. To assess the risk of secondary cancers, comorbidities, mortality and cause-specific mortality in patients with MF treated with nitrogen mustard compared with patients not receiving this treatment. Linking the Danish nationwide registries in a 30-year population-based cohort study, we compared 110 patients with MF from a regional Danish centre using nitrogen mustard treatment with 193 patients from Danish centres not using nitrogen mustard. The two cohorts were compared by Cox regression analysis. Overall, secondary cancers were not significantly increased [hazard ratio (HR) 0.84, 95% confidence interval (CI) 0.46-1.56], and subanalyses showed no significantly increased risk of nonmelanoma skin cancers, malignant melanomas or cancers in the respiratory organs in the nitrogen mustard-treated cohort. Furthermore, we found no significantly increased risk of any category of comorbidity, including chronic pulmonary diseases, in patients treated with nitrogen mustard (HR 0.93, 95% CI 0.48-1.81). Moreover, mortality and cause-specific mortality did not significantly differ between the two cohorts. This study does not support any previous suspicion of increased risk of secondary cancers and chronic pulmonary diseases among patients with MF treated with nitrogen mustard. Furthermore, mortality and cause-specific mortality were not influenced by nitrogen mustard treatment. Thus our findings indicate that topical nitrogen mustard is a safe therapy in patients with MF.

Topical nitrogen mustard therapy in patients with Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is characterized by abnormal proliferation and infiltration of Langerhans cells in different organs. The skin is frequently involved either as unisystem or multisystem disease. To review the clinical response and side-effects of nitrogen mustard therapy in LCH in children and adults with unisystem or multisystem disease. This retrospective study includes 10 children and four adults with LCH, treated with nitrogen mustard from 1975 to 2010. The median extent of skin involvement was 46% (range 5-100%). Overall, 13 patients had complete or partial response. Although eight patients achieved a complete response with a median time of 12·3months (range 36 days to 1·9 years), six of these patients ultimately relapsed. One patient, who had unisystem disease limited to the skin, initially showed progression of her cutaneous lesions with nitrogen mustard treatment. Although subsequently the cutaneous lesions completely regressed, concomitant systemic involvement was noted. Four other patients similarly experienced improvement of their skin lesions with treatment, but also exhibited progression of the LCH systemically. The patients were treated with other therapies prior and adjunctive to nitrogen mustard. However, five patients had progression to other organs, despite regression of skin lesions, which supports that the treatment effect in the skin is related to topical nitrogen mustard. Six patients developed contact dermatitis to nitrogen mustard. Topical nitrogen mustard can be an effective and safe therapy in both children and adults with cutaneous LCH, although relapses are common.

Secondary transitional cell carcinoma and nitrogen mustard treatment

Transitional cell carcinoma (TCC) of the bladder in children is a rare occurrence. Cyclophosphamide is a known risk factor for the development of TCC. Other alkylating agents, such as nitrogen mustard, have not been implicated in the development of secondary adult or pediatric TCC. The role of radiotherapy in the development of secondary malignancies of the bladder remains controversial. We report a case of childhood TCC in a patient in remission from Hodgkin’s lymphoma previously treated with non-cyclophosphamide chemotherapy and low-dose nodal radiotherapy.

Study of ribosome structure using the biochemical methods: judgment day

The data on RNA-RNA interactions between the components of the E. coli translation machinery obtained by X-ray crystallography and chemical methods are compared. The approaches to the study of RNA secondary and tertiary structure are assessed. The following conclusions are made: comparative sequence analysis and compensatory mutations approach both give reliable data on the RNA secondary structure. The chemical modification technique provides good results. Local cleavage of internucleotide bonds by hydroxyl radicals is reliable in the frame of its 40 A resolution, in contrast to the application of copper-phenanthroline complex as a cleavage reagent, which is unreliable. Direct UV irradiation and nitrogen mustard treatment are the best methods of crosslink generation. In vitro transcription is the only good method for the incorporation of nucleotide analogs in RNA. RNase H hydrolysis and/or nucleotide-specific RNases fingerprints must be applied for the crosslink site determination in parallel with reverse transcription.

Chemical Methods for the Structural Study of the Ribosome: Judgment Day

The data on RNA–RNA interactions between the components of the E. colitranslation machinery obtained by X-ray crystallography and chemical methods are compared. The approaches to the study of RNA secondary and tertiary structure are assessed. The following conclusions are made: comparative sequence analysis and compensatory mutations approach both give reliable data on the RNA secondary structure. The chemical modification technique provides good results. Local cleavage of internucleotide bonds by hydroxyl radicals is reliable in the frame of its 40 A resolution, in contrast to the application of copper–phenanthroline complex as a cleavage reagent, which is unreliable. Direct UV irradiation and nitrogen mustard treatment are the best methods of crosslink generation. In vitrotranscription is the only good method for the incorporation of nucleotide analogs in RNA. RNase H hydrolysis and/or nucleotide-specific RNases fingerprints must be applied for the crosslink site determination in parallel with reverse transcription.

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.

Role of the cdc25C phosphatase in G2 arrest induced by nitrogen mustard.

G2 arrest induced by nitrogen mustard in human lymphoma CA46 cells is associated with a failure to activate hyperphosphorylated cdc2/cyclin B1 complexes. We investigated the possibility that this might be due to a suppression of cdc25C phosphatase activity. cdc25C from interphase cells migrated as a 54- to 57-kDa doublet in SDS gels and exhibited basal phosphatase activity. cdc25C from mitotic cells migrated as a 66-kDa hyperphosphorylated species and exhibited elevated phosphatase activity. cdc25C hyperphosphorylation and activation were mediated by cdc2, supporting the view of a cdc2-cdc25C autocatalytic feedback loop. Immunofluorescence and cell fractionation studies suggested cdc2-cdc25C interaction occurred within the cytoplasm. Cells arrested in G2 phase following nitrogen mustard treatment or cells arrested in S phase with aphidicolin failed to dephosphorylate and activate cdc2, and this correlated with failure to convert cdc25C into the most active hyperphosphorylated species. Our findings suggest that checkpoints guarding against mitotic entry in the presence of unreplicated or damaged DNA suppress formation of the cdc2-cdc25C autocatalytic feedback loop that normally brings about rapid activation of cdc2.

Glutathione levels and chemosensitizing effects of buthionine sulfoximine in human malignant glioma cells

Biopsy samples and cultured cells derived from them were obtained from 39 patients with malignant glioma and were analyzed for 1) glutathione (GSH) content; 2) sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and/or nitrogen mustard (HN2) treatment and 3) the effect of buthionine sulfoximine (BSO) treatment on BCNU and/or HN2 cytotoxicity. The average GSH concentration of biopsy specimens was lower than those of cultured cells (2.36 +/- 0.44 vs. 11.42 +/- 2.32 nmol/10(6) cells). While some of the tumor specimens were sensitive to either BCNU or HN2, the majority were resistant to both. However, 8 of 23 tumors tested showed enhanced sensitivity to BCNU following treatment with BSO. Five of 17 tumors were similarly sensitized to HN2 by BSO. These results suggest that BSO chemosensitization may be of value for certain patients and that screening assays may help identify treatment-sensitive individuals.

The role of leucocytes in the induction of fluid secretion by Salmonella typhimurium

Nitrogen mustard (N2M) treatment of rabbits induced neutropenia, and, in ligated ileal loops, it inhibited fluid secretion induced by salmonella or by cholera toxin (CT). Pretreatment of rabbits with indomethacin almost abolished salmonella-induced fluid secretion and significantly reduced that induced by CT. Similar effects of N2M and indomethacin on fluid secretion induced by salmonella, but not by CT, have been reported by other workers and used to implicate prostaglandins, from the salmonella-induced inflammation, as mediators of fluid secretion. In contrast, we show that N2M treatment, in addition to reducing CT-induced secretion, caused severe morphological alterations to ileal mucosa. Irradiation techniques were developed for inducing neutropenia, but they did not totally inhibit salmonella-induced leucocyte influx into ileal mucosa. We propose an alternative mechanism for the inhibitory effect of N2M on salmonella- and CT-induced secretion, based on the known anti-mitotic activity of N2M. Also, the anti-secretory effect of indomethacin cannot be attributed uniquely to its anti-inflammatory activity because it depressed CT-induced secretion as well as salmonella-induced secretion. These results support the concept of pathophysiological secretion in infectious diarrhoea, developed previously for rotavirus and extended to bacterial infections.

Leucocyte infiltration and cartilage proteoglycan loss in immune arthritis in the rabbit

1. The relationship between phagocytic leucocyte infiltration and cartilage degradation in immune arthritis has been investigated in groups of normal and neutropenic rabbits. 2. Injection of antigen into the knee joints of sensitized control animals induced joint swelling, prostaglandin E2 (PGE2) synthesis, leucocyte accumulation and proteoglycan loss from articular cartilage. 3. Intravenous injection of nitrogen mustard caused a selective depletion of circulating neutrophils and monocytes with little or no effect on platelets or lymphocytes. In neutropenic animals challenged with antigen, there was virtually no joint swelling, PGE2 synthesis or leucocyte infiltration but cartilage proteoglycan loss was unchanged after 1 day and increased by day 4 compared to control animals. 4. The numbers of circulating leucocytes returned to normal 3-4 days after nitrogen mustard treatment and leucocyte infiltration occurred in antigen-challenged joints but this was not accompanied by joint swelling. Subsequent intra-articular injection of PGE2 did, however, cause swelling. 5. Lysosomal enzyme levels in arthritic joint fluids were measured. The levels of beta-glucuronidase, which is released by activated phagocytes, were decreased in neutropenic animals but the levels of N-acetyl-beta-glucosaminidase, which is a marker of tissue damage, were not changed by neutrophil depletion. 6. Intra-articular injections of the cytokine interleukin-1 (IL-1) induced a pattern of leucocyte infiltration and cartilage proteoglycan loss similar to that seen in immune arthritis. In neutropenic animals, IL-1 did not cause significant accumulation of leucocytes in the joint but the loss of proteoglycan from cartilage was unimpaired. 7. These results indicate that both leucocyte infiltration and prostaglandin synthesis are required for joint swelling but that tissue degradation is mediated by resident cells. It is likely that release of IL-1 by synovial cells stimulates the synthesis and activation of metalloproteinases which initiate the process of tissue degradation.

Consequences of cell death after nitrogen mustard treatment on skeletal pelvic fin morphogenesis in the trout, Salmo gairdneri (Pisces, teleostei)

Five concentrations of nitrogen mustard (methyl-bis-chlorethylamine) have been administered to different stages of development of trout embryos (Salmo gairdneri), in order to study their effect on the formation of both pelvic fins soft tissue and skeleton. Sensitivity of epidermis and mesoderm toward this cytotoxic agent has been assessed histologically. Although both components are sensitive to nitrogen mustard, mesoderm is more sensitive than epidermis. These results are compared with the results of similar studies on tetrapod limb buds. The pelvic fin skeleton of some treated animals was doubly stained in toto for cartilage and bone by Alcian blue and alizarin, followed by clearing. Other specimens were processed for histological serial sections. Analysis included comparison of the following parameters: (1) dose of nitrogen mustard, (2) stage of fin bud development at the time of treatment, (3) importance of necrosis in the fin bud, and (4) abnormalities of the endoskeleton of the fin (pelvic girdle and radials) and of the dermal skeleton (lepidotrichia and actinotrichia) one month after the hatching of control animals. Resuls suggest (1) that material of endoskeleton and lepidotrichia are laid down simultaneously, and (2) that differentiation of actinotrichia is independent of differentiation in endoskeleton and lepidotrichia.

Induction of tinea cruris by topical nitrogen mustard and by systemic chemotherapy

The present report describes the rapid clinical appearance of superficial fungus infections after the initiation of topical nitrogen mustard treatment in one patient and a short course of systemic cytotoxic drugs in another.

Combined nitrogen mustard-radiation studies with a mouse tumor

The KHT sarcoma, growing in C3H mice, has been treated with nitrogen mustard and irradiation and the response studied as a function of time between the treatments. Two series of experiments have been done and they indicate that a greater response is observed if nitrogen mustard is given before irradiation than if it is given a few hours afterwards. A detailed analysis of the first series of experiments indicated that reoxygenation could occur after drug treatment (within 1 hour) and that at the dose level used (0.2 mg/ mouse) nitrogen mustard treatment preferentially spared the hypoxic cells. Such an analysis has not been completed for the second series of experiments but there are some definite differences between the two groups of experiments, which seem to be due to a change in the response of the tumor to treatment with nitrogen mustard.

Importance of the intestinal inflammatory reaction in salmonella-mediated intestinal secretion

The ability of Salmonella typhimurium to invade the intestinal epithelium is essential to the pathogenesis of salmonella-induced intestinal secretion. This invasion is accompanied by an intense acute inflammatory reaction. The present study tests the hypothesis that the acute inflammatory reaction may have a role in the pathogenesis of salmonella-induced secretion. Two groups of rabbits infected with S. typhimurium were studied: normal animals and animals pretreated with nitrogen mustard. Nitrogen mustard depletes the polymorphonuclear leukocyte pool and thereby prevents the formation of an acute inflammatory reaction. In vivo ligated ileal loops were constructed and infected 72 h after nitrogen mustard administration when polymorphonuclear leukocytes were undetectable. Nitrogen mustard treatment markedly inhibited salmonella-induced secretion. Ileal histology in normal animals infected with S. typhimurium revealed an intense acute inflammatory reaction, while in animals pretreated with nitrogen mustard only a rare polymorphonuclear leukocyte was seen. The antisecretory effect of nitrogen mustard was not merely a nonspecific effect since nitrogen mustard treatment did not inhibit cholera toxin-induced secretion and did not alter either ileal morphology nor the activities of various intestinal enzymes in normal animals. Nitrogen mustard also did not alter the virulence of the inoculated S. typhimurium. These data suggest that the mucosal inflammatory reaction induced by salmonella invasion may be important to the pathogenesis of the salmonella secretory process. The mechanism by which the inflammatory reaction stimulates secretion is not known.

A New Method of Determining the Fraction of Hypoxic Cells in a Transplantable Murine Sarcoma

HILL, R. P., AND BUSH, R. S. A New Method of Determining the Fraction of Hypoxic Cells in a Transplantable Murine Sarcoma. Radiat. Res. 70, 141-153 (1977). Combined nitrogen mustard and radiation treatments of the KHT sarcoma in vivo have indicated that a certain dose of nitrogen mustard can kill all the oxygenated cells in the tumor while killing few, if any, of the hypoxic cells. This finding has allowed nitrogen mustard treatment to be used as a way of selectively removing the oxygenated cells in the tumor so that the radiation response of the hypoxic cells can be studied directly. Using this nitrogen mustard technique the hypoxic fraction is found to be 0.30 as compared to a value of 0.13 obtained using the more conventional technique of comparing anoxic and air-breathing survival data following the same dose of radiation. Also, chronically hypoxic cells have been found to have a smaller shoulder on their radiation survival curve than acutely hypoxic cells.

The alkylation of hemoglobin S by nitrogen mustard. High resolution proton nuclear magnetic resonance studies.

Sickle cell hemoglobin (Hb S) treated with nitrogen mustard (bis(beta-chloroethyl)methylamine hydrochloride) gives two reaction products, one labile and one stable. After dialysis against buffer solution, the remaining stable product is found to inhibit the polymerization of deoxyhemoglobin S. High resolution proton nuclear magnetic resonance has been used to study the structure and function of this stable product and to investigate the nature of the binding sites of nitrogen mustard to the hemoglobin molecule. The NMR results suggest that the nitrogen mustard treatment of Hb S does not alter the heme environment or the subunit interfaces of the hemoglobin molecule. Moreover, the NMR spectra have also shown that the nitrogen mustard reacts with the beta2 histidines of the hemoglobin molecule and have suggested that several other surface amino acid residues of the hemoglobin molecule are also affected by the nitrogen mustard alkylation. These NMR findings are in good agreement with the data obtained from biochemical studies of nitrogen mustard-treated Hb S. The NMR spectra also indicate that nornitrogen mustard (which is also effective in inhibiting sickling) binds with the hemoglobin molecule in a manner identical with nitrogen mustard. Sulfur mustard, on the other hand, produces no observable changes in the aromatic proton resonances, which is consistent with the fact that it does not inhibit the polymerization of deoxy-Hb S.

Nonspecific resistance to intraocular infection. I. Elicitation of resistance by Neisseria meningitidis.

Both eyes of rabbits whose right eyes had previously been injected with Neisseria meningitidis were injected with Diplococcus pneumoniae. The pneumococci failed to grow in the right eye of most rabbits challenged 2 and 4 days after the injection of the meningococci, but grew well in all left eyes. The "protective" effect was less pronounced in the rabbits challenged on days 6 and 9 and virtually absent when the interval between the two injections was 14 or 22 days. Pneumococci grew equally well in both eyes of rabbits that had received nitrogen mustard treatment prior to the intravitreal injection of meningococci, indicating a possible role for the polymorphonuclear leukocyte in the protective effect.

Repair of alkylation damage in ultraviolet-sensitive (xeroderma pigmentosum) human cells

Repair replication occurs in response to alkylation of DNA in normal human cells and in cells from two patients with the hereditary disease xeroderma pigmentosum (XP). Normal and XP cells perform similar amounts of repair replication after treatment with methyl methanesulfonate (MMS) and N-methyl- N'-nitrosoguanidine (MNNG) indicating that such repair is independent of the biochemical defect in XP (presumably a defect in the initial step of excision repair of UV radiation damage). The results imply that these alkylating agents are similar to ionizing radiation and produce chemical breaks in DNA which then induce repair replication. Repair replication levels in normal and XP cells are similar after nitrogen mustard treatment, but these levels are low and within the capacity of the low residual excision repair in XP cell lines. The chemical mechanisms of nitrogen mustard damage are not resolved in these experiments. At high doses of MMS and MNNG repair replication is inhibited, probably because of enzyme inactivation.

Comparative lethal action of ethyl methanesulfonate, nitrogen mustard, myleran and diepoxybutane on T7 coliphage

AbstractInterstrand crosslinks can be shown in phage T7 DNA after treatment by nitrogen mustard or DL‐diepoxybutane, but not after treatment by myleran. Lethality increases after an ethylmethanesulfonate or myleran treatment; this increase is related to the increase of depurinations. Lethality first increases after the nitrogen mustard treatment, then decreases and finally increases again. Crosslinks account for the whole lethality at the end of the treatment, and the evolution of lethality with time can be related to the evolution of crosslinks; the subsequent increase could be correlated to the appearance of fragmentations. Lethality remains constant after the DL‐diepoxybutane treatment; the number of crosslinks is smaller than the number of dead phages; the disappearance of the crosslinks would compensate the biological effect of the increase of depurinated sites so that the lethality remains constant during the first hours after the end of the treatment. The inactivation of the T7 phage by any of these agents is a one‐hit process.