IND.216: a phase II study of buparlisib and associated biomarkers, raptor and p70S6K, in patients with relapsed and refractory chronic lymphocytic leukemia.

Buparlisib is an orally available pan-Class I PI3K inhibitor, that is more potent than idelalisib in vitro. Its distinct toxicities include hyperglycemia, hypertension, and mood disturbance. IND216 is a single arm phase II trial of buparlisib in Relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL). Fourteen patients were enrolled, 13 were evaluable for response and toxicity. Six of 13 patients had a partial response (46%) with a median duration of response of 15.5 months, all 11 patients with tumor assessment experienced tumor shrinkage. The most common adverse events (≥15%) were hyperglycemia, fatigue, anxiety, and gastrointestinal toxicities; all were < grade 3 except for fatigue. Three patients stopped therapy for alterations in mood. Lower levels of raptor were significantly associated with greater tumor shrinkage, suggesting that raptor could be a biomarker for response. This requires further validation in a larger CLL patient cohort. The clinical activity of buparlisib is comparable to other phosphatidylinositol-3-kinase inhibitors, with a different toxicity profile.Novelty and impactBuparlisib, an oral, pan PI3 kinase inhibitor, is associated with a 46% partial response rate among patients with relapse chronic lymphocytic leukemia (CLL). This is a similar clinical activity to other phosphatidylinositol-3-kinase inhibitors tested. However, buparlisib has a distinct toxicity profile, characterized by hyperglycemia, hypertension, and mood alteration. In agreement with our previous preclinical study, our results suggest that basal raptor expression in CLL correlates with clinical response to buparlisib.

PARP3 inhibitors ME0328 and olaparib potentiate vinorelbine sensitization in breast cancer cell lines.

PURPOSE: PARP-3 is member of the PARP family of poly (ADP-ribose) polymerases involved in ADPribosylation. PARPs are involved in the basic mechanisms of DNA repair. PARP3, a critical player for efficient mitotic progression, is required for the stabilization of the mitotic spindle by regulation of the mitotic components, NuMA and Tankyrase 1. METHODS: The sensitization effect of vinorelbine on PARP3 inhibition-induced cytotoxicity was assessed by the SRB assay. The contribution of programed cell death and cell cycle arrest to the sensitization effect were determined by assessing changes in Annexin V, a marker of apoptosis. Alterations in cell cycle progression were assessed by cell cycle analysis. We used immunofluorescence to assess the effect of vinorelbine and/or PARP3 inhibitors on tubulin and microtubule depolarization. The PARP3 chemiluminescent assay kit was used for PARP3 activity. RESULTS: PARP3 inhibitors sensitize breast cancer cells to vinorelbine, a vinca alkaloid used in the treatment of metastatic breast cancer. Olaparib which was originally described as a PARP1 and 2 inhibitor has recently been shown to be a potent PARP3 inhibitor while ME0328 is a more selective PARP3 inhibitor. The combination of vinorelbine with nontoxic concentrations of ME0328 or olaparib reduces vinorelbine resistance by 10 and 17 fold, respectively, potentiating vinorelbine-induced arrest at the G2/M boundary. In addition, PARP3 inhibition potentiates vinorelbine interaction with tubulin. Furthermore, olaparib or ME0328 potentiates vinorelbine-induced PARP3 inhibition, mitotic arrest, and apoptosis. CONCLUSION: Our results indicated this approach with PARP3 inhibitors and vinorelbine is unique and promising for breast cancer patients with metastases. This combination could significantly increase the survival of breast cancer patients with metastases.

Precision spherical nucleic acids for delivery of anticancer drugs.

We report a spherical nucleic acid (SNA) system for the delivery of BKM120, an anticancer drug for treatment of chronic lymphocytic leukemia (CLL). While promising for cancer treatment, this drug crosses the blood-brain barrier causing significant side-effects in patients. The DNA nanoparticle encapsulates BKM120 in high efficiency, and is unparalleled in its monodispersity, ease of synthesis and stability in different biological media and in serum. These DNA nanostructures demonstrate efficient uptake in human cervical cancer (HeLa) cells, and increased internalization of cargo. In vitro studies show that BKM120-loaded nanoparticles promote apoptosis in primary patient CLL lymphocytes, and act as sensitizers of other antitumor drugs, without causing non-specific inflammation. Evaluation of this drug delivery system in vivo shows long circulation times up to 24 hours, full body distribution, accumulation at tumor sites and minimal leakage through the blood-brain barrier. Our results demonstrate the great potential of these delivery vehicles as a general platform for chemotherapeutic drug delivery.

Simultaneous inhibition of ATR and PARP sensitizes colon cancer cell lines to irinotecan.

Enhanced DNA damage repair is one mechanism involved in colon cancer drug resistance. Thus, targeting molecular components of repair pathways with specific small molecule inhibitors may improve the efficacy of chemotherapy. ABT-888 and VE-821, inhibitors of poly-ADP-ribose-polymerase (PARP) and the serine/threonine-kinase Ataxia telangiectasia related (ATR), respectively, were used to treat colon cancer cell lines in combination with the topoisomerase-I inhibitor irinotecan (SN38). Our findings show that each of these DNA repair inhibitors utilized alone at nontoxic single agent concentrations resulted in sensitization to SN38 producing a 1.4-3 fold reduction in the 50% inhibitory concentration (IC50) of SN38 in three colon cancer cell lines. When combined together, nontoxic concentrations of ABT-888 and VE-821 produced a 4.5-27 fold reduction in the IC50 of SN38 with the HCT-116 colon cancer cells demonstrating the highest sensitization as compared to LoVo and HT-29 colon cancer cells. Furthermore, the combination of all three agents was associated with maximal G2 -M arrest and enhanced DNA-damage (γH2AX) in all three colon cancer cell lines. The mechanism of this enhanced sensitization was associated with: (a) maximal suppression of SN38 induced PARP activity in the presence of both inhibitors and (b) ABT-888 producing partial abrogation of the VE-821 enhancement of SN38 induced DNA-PK phosphorylation, resulting in more unrepaired DNA damage; these alterations were only present in the HCT-116 cells which have reduced levels of ATM. This novel combination of DNA repair inhibitors may be useful to enhance the activity of DNA damaging chemotherapies such as irinotecan and help produce sensitization to this drug in colon cancer.

Small Molecules, Inhibitors of DNA-PK, Targeting DNA Repair, and Beyond.

Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining a major mechanism for the repair of double-strand breaks (DSB) in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK). The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its' central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA-PK. Computer based drug design will not only assist in identifying novel functional moieties to replace the metabolically labile morpholino group but will also facilitate the design of molecules to target the DNA-PKcs/Ku80 interface or one of the autophosphorylation sites.

Heterozygous mutations in the PALB2 hereditary breast cancer predisposition gene impact on the three-dimensional nuclear organization of patient-derived cell lines.

PALB2/FANCN is a BRCA1- and BRCA2-interacting Fanconi Anemia (FA) protein crucial for key BRCA2 genome caretaker functions. Heterozygous germline mutations in PALB2 predispose to breast cancer and biallelic mutations cause FA. FA proteins play a critical role in the telomere maintenance pathway, with telomeric shortening observed in FA cells. Less is known about telomere maintenance in the heterozygous state. Here, we investigate the roles of PALB2 heterozygous mutations in genomic instability, an important carcinogenesis precursor. Patient-derived lymphoblastoid (LCL) and fibroblast (FCL) cell lines with monoallelic truncating PALB2 mutations were investigated using a combination of molecular imaging techniques including centromeric FISH, telomeric Q-FISH and spectral karyotyping (SKY). Mitomycin C and Cisplatin sensitivity was assayed via cellular metabolism of WST-1. The PALB2 c.229delT FCL showed increases in telomere counts associated with increased mean intensity compared with two wild-type FCLs generated from first-degree relatives (P =1.04E-10 and P =9.68E-15) and it showed evidence of chromosomal rearrangements. Significant differences in centromere distribution were observed in one of three PALB2 heterozygous FCLs analyzed when compared with PALB2 wild-type, BRCA1 and BRCA2 heterozygous FCLs. No significant consistently increased sensitivity to Mitomycin C or Cisplatin was observed in LCLs. Our results are suggestive of an altered centromere distribution profile and a telomere instability phenotype. Together, these may indicate critical nuclear organization defects associated with the predisposition to transformation and early stage development of PALB2-related cancers.

The phosphatidylinositol-3 kinase I inhibitor BKM120 induces cell death in B-chronic lymphocytic leukemia cells in vitro.

BKM120, a pan class I PI3K inhibitor, was cytotoxic in the majority of primary B-chronic lymphocytic leukemia (CLL) lymphocytes, including samples from patients who have a high-risk for poor response to treatment (patient with del11 and del17) at clinically obtainable concentrations. The PI3Kδ inhibitor Cal-101 is cytotoxic in B-CLL lymphocytes in vitro and is active in the treatment of CLL in vivo. Interestingly, we demonstrated that BKM120 is 3.6 fold more toxic than Cal-101 in malignant B-CLL lymphocytes in vitro. BKM120 cytotoxicity correlated with the basal expression of proteins involved in the PI3K/Akt pathway. A protein signature of PI3K pathway proteins predicts the response to BKM120 treatment. In the primary B-CLL lymphocytes tested in vitro, BKM120 decreased the phosphorylation status of molecular biomarkers used as indicators of PI3K pathway inhibition in vivo. Also, BKM120 induced apoptosis in primary B-CLL cells culture in the presence and absence of stromal cell support. Our findings suggest that BKM120 should be tested clinically in CLL.

Metformin reduces endogenous reactive oxygen species and associated DNA damage.

Pharmacoepidemiologic studies provide evidence that use of metformin, a drug commonly prescribed for type II diabetes, is associated with a substantial reduction in cancer risk. Experimental models show that metformin inhibits the growth of certain neoplasms by cell autonomous mechanisms such as activation of AMP kinase with secondary inhibition of protein synthesis or by an indirect mechanism involving reduction in gluconeogenesis leading to a decline in insulin levels and reduced proliferation of insulin-responsive cancers. Here, we show that metformin attenuates paraquat-induced elevations in reactive oxygen species (ROS), and related DNA damage and mutations, but has no effect on similar changes induced by H(2)0(2), indicating a reduction in endogenous ROS production. Importantly, metformin also inhibited Ras-induced ROS production and DNA damage. Our results reveal previously unrecognized inhibitory effects of metformin on ROS production and somatic cell mutation, providing a novel mechanism for the reduction in cancer risk reported to be associated with exposure to this drug.

Effects of the novel DNA dependent protein kinase inhibitor, IC486241, on the DNA damage response to doxorubicin and cisplatin in breast cancer cells.

The purpose of this study was to determine the degree to which the novel DNA-PKcs inhibitor, IC486241 (ICC), synergizes the cytotoxicity of DNA damaging agents in 3 genetically diverse breast cancer cell lines. The sulforhodamine B (SRB) assay was employed as a primary screening method to determine the in-vitro cytotoxicity and the degree of synergy of ICC in combination with the topoisomerase II inhibitor, doxorubicin, or the DNA cross linking agent, cisplatin. Molecular mechanisms underlying drug toxicity were probed using immunostaining and flow cytometry, as well as, the alkaline comet assay to detect DNA damage. In this study, improved cytotoxicity and significant synergy were observed with both anticancer agents in the presence of nontoxic concentrations of ICC. Moreover, ICC decreased doxorubicin-induced DNA-PKcs autophosphorylation on Ser2056 and increased doxorubicin-induced DNA fragmentation. In conclusion, the novel DNA-PKcs inhibitor, ICC, synergistically sensitized 3 breast cancer cell lines to doxorubicin and cisplatin. Enhanced efficacy of doxorubicin was achieved by inhibiting non-homologous end joining resulting in increased accumulation of DNA damage.

Irinotecan and DNA-PKcs inhibitors synergize in killing of colon cancer cells.

This study sought to measure the degree of synergy induced by specific small molecule inhibitors of DNA-PK [NU7026 and IC486241 (ICC)], a major component of the non-homologous end-joining (NHEJ) pathway, with SN38 or oxaliplatin. Synergy between the DNA damaging drugs and the DNA-PK inhibitors was assessed using the sulforhodamine-B assay (SRB). Effects of drug combinations on cell cycle and DNA-PK activity were determined using flow cytometry and western blot analysis. DNA damage was assessed via comet assay and quantification of γH2AX. The role of homologous recombination repair (HRR) was determined by nuclear Rad51 protein levels and a GFP reporter recombination assay. Significant reductions in the IC(50) values of SN38 were observed at 5 and 10 µM of DNA-PK inhibitors. Moreover, at 1-2 µM (attainable concentrations with ICC in mice) these DNA-PKcs inhibitors demonstrated synergistic reductions in the IC(50) of SN38. Flow cytometric data indicated that SN38 and SN38 in combination with DNA-PKcs inhibitors showed dramatic G2/M arrest at 24 h. Furthermore, reduced phosphorylation of DNA-PKcs and increased DNA damage were observed at this time point with SN38 in combination with DNA-PKcs inhibitors as compared to cells treated with SN38 alone. SN38 alone and in the presence of ICC increased nuclear Rad51 protein levels. Furthermore, inhibition of DNA-PKcs increased HRR suggesting that NHEJ is a negative regulator of HRR. These data indicate that small molecule inhibitors of DNA-PKcs dramatically enhance the efficacy of SN38 in colon cancer cell lines.

Dual inhibition of the homologous recombinational repair and the nonhomologous end-joining repair pathways in chronic lymphocytic leukemia therapy.

Resistance to chlorambucil in chronic lymphocytic leukemia (CLL) has been associated with increased DNA repair. Specifically, inhibition of either c-abl, which modulates Rad51 directed homologous recombination or DNA-PK dependent nonhomologous end joining has been shown to sensitize primary CLL lymphocytes to chlorambucil. Here we report that inhibition of c-abl can result in a compensatory increase in DNA-PK and thus inhibition of both c-abl and DNA-PK optimally sensitizes CLL lymphocytes to chlorambucil. In this paper we report a drug-induced compensatory change between two DNA repair pathways with potential therapeutic implications in CLL therapy.

p53 and autophagy contribute to dasatinib resistance in primary CLL lymphocytes.

B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in adults and there is no cure for the disease. Although dasatinib is cytotoxic to primary CLL lymphocytes in vitro, the drug has been shown to be active in a small percent of CLL patients. Our previous results suggest that dasatinib targets del17 CLL lymphocytes which are the CLL patients with the worst prognosis. Here we present mechanistic evidence that dasatinib induces endoplasmic reticulum stress and autophagy in CLL lymphocytes. Furthermore we provide evidence suggesting that autophagy mediates resistance to the drugs, process that is modulated by p53.

A phase I study of imatinib mesylate in combination with chlorambucil in previously treated chronic lymphocytic leukemia patients.

PURPOSE: The tyrosine kinase inhibitor, imatinib, has the potential to indirectly inhibit DNA repair. This mechanism of action has been shown to mediate sensitization to chlorambucil in chronic lymphocytic leukemia (CLL). To evaluate this effect in vivo, we performed a phase I study of chlorambucil combined with imatinib in relapsed CLL patients. METHODS: The three dose levels studied included imatinib at 300, 400, or 600 mg/day. Imatinib was given on days 1-10, and chlorambucil (8 mg/m(2) daily) was given on days 3-7 of a 28-day cycle (up to 6 cycles). RESULTS: Eleven patients participated in this study. Low-grade gastrointestinal toxicities were observed in a dose-dependent manner. Forty-five percent of patients responded (two unconfirmed CRs and three PRs). Two responding patients were fludarabine refractory. The in vitro IC(50) of chlorambucil alone or in the presence of 5 µM imatinib in CLL lymphocytes correlated with the decrease in lymphocyte counts on day 15. Imatinib plasma concentrations achieved in patients were in the range of those effective in in vitro sensitization studies. CONCLUSION: The combination of chlorambucil and imatinib in patients with previously treated CLL was well tolerated and showed evidence of clinical efficacy. Based on our results, we recommend the 400 mg daily dose of imatinib on days 1-10 with 8 mg/m(3) chlorambucil on days 3-7 every 28 days as the phase II dose. This represents the first clinical trial examining the potential synergy between a tyrosine kinase inhibitor and a conventional alkylating agent for the treatment of CLL.

Metformin blocks the stimulative effect of a high-energy diet on colon carcinoma growth in vivo and is associated with reduced expression of fatty acid synthase.

The molecular mechanisms responsible for the association of obesity with adverse colon cancer outcomes are poorly understood. We investigated the effects of a high-energy diet on growth of an in vivo colon cancer model. Seventeen days following the injection of 5x10(5) MC38 colon carcinoma cells, tumors from mice on the high-energy diet were approximately twice the volume of those of mice on the control diet. These findings were correlated with the observation that the high-energy diet led to elevated insulin levels, phosphorylated AKT, and increased expression of fatty acid synthase (FASN) by the tumor cells. Metformin, an antidiabetic drug, leads to the activation of AMPK and is currently under investigation for its antineoplastic activity. We observed that metformin blocked the effect of the high-energy diet on tumor growth, reduced insulin levels, and attenuated the effect of diet on phosphorylation of AKT and expression of FASN. Furthermore, the administration of metformin led to the activation of AMPK, the inhibitory phosphorylation of acetyl-CoA carboxylase, the upregulation of BNIP3 and increased apoptosis as estimated by poly (ADP-ribose) polymerase (PARP) cleavage. Prior work showed that activating mutations of PI3K are associated with increased AKT activation and adverse outcome in colon cancer; our results demonstrate that the aggressive tumor behavior associated with a high-energy diet has similar effects on this signaling pathway. Furthermore, metformin is demonstrated to reverse the effects of the high-energy diet, thus suggesting a potential role for this agent in the management of a metabolically defined subset of colon cancers.

Dasatinib sensitizes primary chronic lymphocytic leukaemia lymphocytes to chlorambucil and fludarabine in vitro.

The dual c-abl/Src kinase inhibitor, dasatinib, utilized to treat chronic myeloid leukaemia (CML) when used at clinically attainable sublethal concentrations, synergistically sensitized primary chronic lymphocytic leukaemia (CLL) lymphocytes to chlorambucil and fludarabine. In contrast, dasatinib alone demonstrated toxicity to CLL lymphocytes at concentrations that are generally not clinically attainable. Dasatinib resistance and poorer dasatinib-mediated sensitization to chlorambucil and fludarabine was associated with higher expression of c-abl protein levels. In contrast, chlorambucil and fludarabine resistance correlated with basal p53 protein levels. Moreover, Western blot analysis after in vitro treatment of primary CLL lymphocytes with dasatinib, chlorambucil and/or fludarabine, showed that dasatinib: (i) inhibited c-abl function (e.g. downregulation of c-abl protein levels and decreased the phosphorylation of a c-abl downstream target, Dok2), (ii) decreased chlorambucil/fludarabine induced accumulation of p53 protein levels, (iii) altered the response to chlorambucil/fludarabine induced DNA-damage as evidenced by an increase in chlorambucil/fludarabine-induced H2AX phosphorylation, and (iv) accentuated the c-abl downregulation induced by chlorambucil/fludarabine. Our results suggest that dasatinib in combination with chlorambucil or fludarabine may improve the therapy of CLL.

Chlorambucil cytotoxicity in malignant B lymphocytes is synergistically increased by 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026)-mediated inhibition of DNA double-strand break repair via inhibition of DNA-dependent protein kinase.

Chlorambucil (CLB) treatment is used in chronic lymphocytic leukemia (CLL) but resistance to CLB develops in association with accelerated repair of CLB-induced DNA damage. Phosphorylated histone H2AX (gammaH2AX) is located at DNA double-strand break (DSB) sites; furthermore, it recruits and retains damage-responsive proteins. This damage can be repaired by nonhomologous DNA end-joining (NHEJ) and/or homologous recombinational repair (HR) pathways. A key component of NHEJ is the DNA-dependent protein kinase (DNA-PK) complex. Increased DNA-PK activity is associated with resistance to CLB in CLL. We used the specific DNA-PK inhibitor 2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026) to sensitize CLL cells to chlorambucil. Our results indicate that in a CLL cell line (I83) and in primary CLL-lymphocytes, chlorambucil plus NU7026 has synergistic cytotoxic activity at nontoxic doses of NU7026. CLB treatment results in G(2)/M phase arrest, and NU7026 increases this CLB-induced G(2)/M arrest. Moreover, a kinetic time course demonstrates that CLB-induced DNA-PK activity was inhibited by NU7026, providing direct evidence of the ability of NU7026 to inhibit DNA-PK function. DSBs, visualized as gammaH2AX, were enhanced 24 to 48 h after CLB and further increased by CLB plus NU7026, suggesting that the synergy of the combination is mediated by NU7026 inhibition of DNA-PK with subsequent inhibition of DSB repair.

XRCC3 depletion induces spontaneous DNA breaks and p53-dependent cell death.

In vertebrate cells, Xrcc3 initiates the repair of exogenous induced-DNA breaks during S and G(2)/M phases of the cell cycle by homologous recombination. However, much less is known of the role of Xrcc3 in the response to spontaneous DNA breaks. Using a siRNA approach, we show that depletion of XRCC3 inhibits the proliferation of MCF7 breast cancer cells. This inhibition of replication coincides with the accumulation of DNA breaks, as shown by the comet assay. Cell cycle specific analysis of gammaH2AX expression shows that S and G2/M phase cells express the highest fraction of gammaH2AX positive cells. This is consistent with replication-dependent accumulation of DNA breaks and deficient homologous recombination. While the induction of gammaH2AX is followed by cell death in parental cells, a p53 knockdown derivative becomes more resistant to XRCC3 depletion-induced death without changes in the levels of gammaH2AX. These results show that XRCC3 is required for the proliferation of MCF7 cells, and that decrease in its expression leads to the accumulation of DNA breaks and the induction of p53-dependent cell death.

Expression patterns of nm23 genes during mouse organogenesis.

Nucleoside di-phosphate kinase enzyme (NDPK) isoforms, encoded by the nm23 family of genes, may be involved in various cellular differentiation and proliferation processes. We have therefore analyzed the expression of nm23-M1, -M2, -M3, and -M4 during embryonic mouse development. In situ hybridization data has revealed the differential expression of nm23 mRNA during organogenesis. Whereas nm23-M1 and -M3 are preferentially expressed in the nervous and sensory systems, nm23-M2 mRNA is found ubiquitously. Irrespective of the developmental state studied, nm23-M4 mRNA is only expressed at low levels in a few embryonic organs. In the cerebellum and cerebral cortex, nm23-M1, -M2, and -M3 are present in the neuronal differentiation layer, whereas nm23-M4 mRNA is distributed in the proliferating layer. Thus, nm23 mRNA is differentially expressed, and the diverse NDPK isoforms are sequentially involved in various developmental processes.

Differential expression of nm23 genes in adult mouse dorsal root ganglia.

Nm23 has been identified as a gene family encoding different isoforms of nucleoside diphosphate kinase (NDPK). This protein is a key enzyme in nucleotide metabolism and has been shown to play important roles in various cellular functions. In the present study, we have investigated the expression of three isotypes in mouse dorsal root ganglia. In situ hybridization and reverse transcriptase-polymerase chain reaction analysis demonstrated high levels of nm23-M1, -M2, and -M3 mRNA expression in peripheral nervous tissue. Moreover, in situ hybridization also displayed a specific nuclear localization for nm23-M2 mRNA. Immunohistochemistry with light and electron microscopy on isoform-specific antibodies revealed a differential subcellular distribution of NDPK isoforms. Isoform A was mainly cytosolic, showing only partial association with organelles. In contrast, isoform B was also found in the nucleus, which is in agreement with its proposed role as a transcription factor. The results also indicate a preferential association of isoform C with endoplasmic reticulum and plasma membranes in neuronal cells. Furthermore, isoform C appeared to combine with other NDPK isoforms as demonstrated by double-labeling evidence by electron microscopy and might be responsible for binding NDPK oligomers to membranes. Thus, isoform C may be considered as a protein of importance for maintaining intracellular pools of GTP in the vicinity of membranes and, hence, for transmembrane signaling. The results indicate a high expression of NDPK isoforms, not only in the central but also in the peripheral nervous system. Their different subcellular compartmentalization suggests that they have isoform-specific roles in neuronal cell physiology.