DNA Synthesis In Tumor Cells Research Articles

New therapeutic strategies based on interference with telomeric DNA synthesis of tumor cells to suppress the growth of tumors.

An unusual enzyme called telomerase acts on parts of chromosomes known as telomeres. The enzyme has recently been found in many human tumors and is viewed as a new target for tumor therapy. In this research, we chose the analogue of guanine “2′,3′-dideoxyguanosine” (ddG) as the telomerase inhibitor and prepared the ddG-loaded cationic nanoliposomes (ddG-Clip) to specifically target the tumor tissue and preferentially occupy the telomerase nucleotide binding site. The mean diameter of ddG-Clip is 101.54 ± 2.60 nm and they are cationically charged with a zeta potential of 34.0 ± 9.43 mV; also, the encapsulation efficiency of ddG-Clip is 53.44% ± 2.29%. In vitro cytotoxicity results show that cationic nanoliposomes by themselves are almost non-toxic, but with the increase in ddG concentration, ddG-Clip has the ability to kill S180 tumor cells. The anti-tumor activity study suggests that ddG-Clip could not only suppress the tumor growth, but also inhibit tumor liver metastasis well. In conclusion, reverse transcriptase inhibitor-loaded cationic nanoliposomes could interfere with the synthesis of telomeric DNA and block abnormal proliferation of tumor cells, therefore achieving tumor apoptosis.

Digalloylresveratrol, a novel resveratrol analog inhibits the growth of human pancreatic cancer cells

Digalloylresveratrol (DIG) is a recently synthesized substance aimed to combine the effects of the natural polyphenolic compounds gallic acid and resveratrol, which both are excellent free radical scavengers with anticancer activity. In this study, we investigated the effects of DIG in the human AsPC-1 and BxPC-3 pancreatic adenocarcinoma cell lines. Treatment with DIG dose-dependently attenuated cells in the S phase of the cell cycle and led to a significant depletion of the dATP pool in AsPC-1 cells. The incorporation of (14)C-cytidine into nascent DNA of tumor cells was significantly inhibited at all DIG concentrations due to inhibition of ribonucleotide reductase, a key enzyme of DNA synthesis in tumor cells. Furthermore, Erk1/2 became inactivated and moderated p38 phosphorylation reflecting increased replication stress. DIG also activated ATM and Chk2, and induced the phosphorylation and proteasomal degradation of the proto-oncogene Cdc25A, which contributed to cell cycle attenuation. Taken together, DIG is an excellent free radical scavenger, strongly inhibits RR in situ activity, cell cycle progression, and colony formation in AsPC-1 and BxPC-3 cells thus warranting further investigations.

Redox-active ferrocene-modified pyrimidines and adenine as antitumor agents: structure, separation of enantiomers, and inhihibition of the DNA synthesis in tumor cells

The structures, electrochemical properties, enantiomeric separation of ferrocenyl-(alkyl)pyrimidines and ferrocenyl(ethyl)adenine and their effects on the DNA synthesis in tumor cells were studied. Enantiomeric mixtures were separated by HPLC on modified cellulose as the chiral selector. The electrochemical properties of compounds were studied by cyclic voltammetry. All compounds have reversible single-electron redox transition in the region of 0.52–0.60 V, which belongs to ferrocene—ferrocenium with a positive shift compared to ferrocene (0.52 V). The molecular structure of 1-N-(ferrocenylbenzyl)-5-iodocytosine was studied by X-ray diffraction. 1-N-(Ferrocenylethyl)adenine was studied for ability to inhibit the DNA synthesis in the human ovarian cancer cell culture by the 3H-thymidine test.

Synthesis of vitamin–selenium complex and its effects on proteins and tumor cells

A selenium–vitamin P complex (SEVP) was synthesized and its structure was determined by IR, LC–MS and 1H NMR. Its biological effects on bovine serum albumin (BSA) and human colon carcinoma tumor cells were studied by molecular spectra, MTT and flow cytometry. The interaction of SEVP and BSA was discussed by fluorescence quenching method and Förster non-radiation energy transfer theory. The thermodynamic parameters Δ H θ , Δ G θ , Δ S θ at different temperatures were calculated and the results indicate the interaction is an exothermic as well as entropy-driven process. Hydrogen bond and electrostatic force played major role in the reaction. The binding geometry and conformation changes of BSA were investigated by fluorescence probe technique and circular dichroism (CD) spectra. The effects of SEVP on human colon carcinoma tumor cells HT29 were tested by MTT method and flow cytometry (FCM). The MTT results show the proliferation of HT29 tumor cells were inhibited by SEVP and the inhibition was associated with dose and time. The FCM analysis disclosed SEVP interrupted the DNA synthesis of tumor cells at S phase.

Error-prone translesion synthesis mediates acquired chemoresistance

The development of cancer drug resistance is a persistent clinical problem limiting the successful treatment of disseminated malignancies. However, the molecular mechanisms by which initially chemoresponsive tumors develop therapeutic resistance remain poorly understood. Error-prone translesional DNA synthesis (TLS) is known to underlie the mutagenic effects of numerous anticancer agents, but little is known as to whether mutation induced by this process is ultimately relevant to tumor drug resistance. Here, we use a tractable mouse model of B-cell lymphoma to interrogate the role of error-prone translesional DNA synthesis in chemotherapy-induced mutation and resistance to front-line chemotherapy. We find that suppression of Rev1, an essential TLS scaffold protein and dCMP transferase, inhibits both cisplatin- and cyclophosphamide-induced mutagenesis. Additionally, by performing repeated cycles of tumor engraftment and treatment, we show that Rev1 plays a critical role in the development of acquired cyclophosphamide resistance. Thus, chemotherapy not only selects for drug-resistant tumor population but also directly promotes the TLS-mediated acquisition of resistance-causing mutations. These data provide an example of an alteration that prevents the acquisition of drug resistance in tumors in vivo. Because TLS also represents a critical mechanism of DNA synthesis in tumor cells following chemotherapy, these data suggest that TLS inhibition may have dual anticancer effects, sensitizing tumors to therapy as well as preventing the emergence of tumor chemoresistance.

Ribonucleotide Reductase as One Important Target of [Tris(1,10- phenanthroline)lanthanum(III)] Trithiocyanate (KP772)

KP772 is a new lanthanum complex containing three 1,10-phenathroline molecules. Recently, we have demonstrated that the promising in vitro and in vivo anticancer properties of KP772 are based on p53-independent G(0)G(1) arrest and apoptosis induction. A National Cancer Institute (NCI) screen revealed significant correlation of KP772 activity with that of the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU). Consequently, this study aimed to investigate whether KP772 targets DNA synthesis in tumor cells by RR inhibition. Indeed, KP772 treatment led to significant reduction of cytidine incorporation paralleled by a decrease of deoxynucleoside triphosphate (dNTP) pools. This strongly indicates disruption of RR activity. Moreover, KP772 protected against oxidative stress, suggesting that this drug might interfere with RR by interaction with the tyrosyl radical in subunit R2. Additionally, several observations (e.g. increase of transferrin receptor expression and protective effect of iron preloading) indicate that KP772 interferes with cellular iron homeostasis. Accordingly, co-incubation of Fe(II) with KP772 led to generation of a coloured iron complex (Fe-KP772) in cell free systems. In electron paramagnetic resonance (EPR) measurements of mouse R2 subunits, KP772 disrupted the tyrosyl radical while Fe-KP772 had no significant effects. Moreover, coincubation of KP772 with iron-loaded R2 led to formation of Fe-KP772 suggesting chelation of RR-bound Fe(II). Summarizing, our data prove that KP772 inhibits RR by targeting the iron centre of the R2 subunit. As also Fe-KP772 as well as free lanthanum exert significant -though less pronounced- cytotoxic/static activities, additional mechanisms are likely to synergise with RR inhibition in the promising anticancer activity of KP772.

The high affinity peroxisome proliferator‐activated receptor‐gamma agonist RS5444 inhibits both initiation and progression of colon tumors in azoxymethane‐treated mice

We evaluated RS5444, a thiazolidinedione high affinity PPARgamma agonist, for the ability to inhibit colon carcinogenesis in azoxymethane (AOM)-treated mice. In our initial experiment, mice were treated with RS5444 during AOM treatment, and the drug was withdrawn 12 weeks after the last injection of AOM. RS5444 significantly inhibited aberrant crypt focus formation under these circumstances. Furthermore, exposure to RS5444 during the course of AOM treatment effectively blocked colon tumor formation after withdrawal of the agonist. PPARgamma expression and nuclear localization were reduced in adenomas. RS5444 did not inhibit DNA synthesis in tumor cells, suggesting that PPARgamma activity was impaired in adenomas. To test this hypothesis, pre-existing adenomas were treated with RS5444 for 16 weeks. We observed a slight, albeit not statistically significant, reduction in tumor incidence in RS5444-treated mice. However, histological examination revealed that tumors from RS5444-treated mice were of significantly lower grade, as evaluated by the extent of dysplasia. Furthermore, carcinoma in situ was observed in about one-third of control tumors, but was never observed in RS5444-treated tumors. We conclude that RS5444 inhibits both initiation and progression of colon tumors in the AOM model of sporadic colon carcinogenesis.

Modulation of Circadian Rhythm of DNA Synthesis in Tumor Cells by Inhibiting Platelet-Derived Growth Factor Signaling

Circadian synchronization of cell proliferation is observed not only in normal healthy tissues but also in malignant solid tumors. However, the proliferation rhythm of tumor cells is often different from that of normal cells. We reported here that the peculiar rhythm of tumor cell proliferation was modulated by inhibition of platelet-derived growth factor (PDGF) signaling. DNA synthesis in tumor cells implanted in mice showed a 24-h oscillation apparently differing from that of normal bone marrow cells. Continuous administration of AG1295 (10 μg/h, s.c.), a PDGF receptor tyrosine kinase inhibitor, substantially suppressed DNA synthesis in the implanted tumor cells but not in the healthy bone marrow cells. During the administration of this drug, the rhythm of DNA synthesis in the tumor cells was synchronized with that in bone marrow cells. The present results suggest that the circadian rhythm of DNA synthesis in tumor cells is modulated by PDGF receptor signaling, which is activated following tumor progression. Because the rhythmic patterns of clock gene expression in tumor cells did not differ significantly from those in other healthy tissues, the enhanced signal transduction of PDGF receptor may cause an alteration in the rhythmicity of tumor cell proliferation without changing in the intracellular molecular clockwork.

The Anti-Tumor Effect and Mechanisms of Action of Penta-Acetyl Geniposide

Gardenia, the fruit of Gardenia jasminoides Ellis, has been widely used to treat liver and gall bladder disorders in Chinese medicine. It has been shown recently that geniposide, the main ingredient of Gardenia Fructus, exhibits the anti-tumor effect. In this review, we discuss the anti-tumor effect and possible mechanisms of a derivative from Gardenia Fructus, penta-acetyl geniposide ((Ac)5GP). It has been demonstrated that (Ac)5GP plays more potent roles than geniposide in chemoprevention. (Ac)5GP decreased DNA damage and hepatocarcinogenesis induced by aflatoxin B1 (AFB1) by activating the phase II enzymes glutathione S-transferase (GST) and GSH peroxidase (GSH-Px). It reduced the growth and development of inoculated C6 glioma cells especially in pre-treated rats. In addition to the preventive effect, (Ac)5GP exerts its actions on apoptosis and growth arrest. Treatment of (Ac)5GP caused DNA fragmentation of glioma cells. (Ac)5GP induced sub- G1 peak through the activation of apoptotic cascades PKCdelta/JNK/Fas/caspase8 and caspase 3. Besides, p53/Bax signaling was suggested to be involved in (Ac)5GP-induced apoptosis, though its downstream cascades needs further clarified. (Ac)5GP has also been shown to inhibit DNA synthesis of tumor cells. It arrested cell cycle at G0/ G1 by inducing the expression of p21, thus suppressing the cyclin D1/cdk4 complex formation and the phosphorylation of E2F. The phosphorylation status of p53 on serine 392 correlated with the process of growth arrest. Evidences from the in vivo experiments showed that (Ac)5GP is not harmful to liver, heart and kidney. In conclusion, (Ac)5GP is highly suggested to be an anti-tumor agent for development in the future.

Immunochemoconjugate of anti-DNMT1/HDAC2 bispecific F(ab)2-bsAb linked with cleavable disulfide to vinorelbine induces apoptosis in chemoresistant infiltrating ductal carcinoma (IDC) characterised by HDAC2 overexpression and 5'CpG island hypermethylation of the FHIT, RAR-β2, BRCA-1, APC, p16(CDKN2A), RASSF1A, CDH1(E-cadherin), 14–3-3-σ (stratifin), HIC1 and MDG1 tumor suppressor genes

2088 One of the reasons for chemoresistance to IDC is deacetylation and DNA methylation which causes silencing of tumor suppressor genes(TSGs).DNMT1 interacts with HDAC2 to repress transcription of TSGs. IDC cells obtained by surgical excision from a patient were analysed by IHC,PCR-based LOH, ChIP, MSP,RT-PCR and NB.There was loss of FHIT expression,LOH at FHIT and 5' CpG island methylation of the FHIT .There was transcriptional silencing of the following TSGs: BRCA1,RAR-β2,APC,p16 (CDKN2A), RASSF1A,CDH1(E-cadherin),14–3-3-σ (stratifin),HIC1 and MDG1.There was overexpression of DNMT1 and HDAC2 suggesting a link between histone deacetylation,cytosine methylation,local chromatin condensation and subsequent transcriptional repression.This chemoresistant IDC was defined as CIMP+.We treated IDC cells with anti-DNMT1/HDAC2 bispecific F(ab)2-bsAb linked with cleavable disulfide to VRL . Post-treatment, there was inhibition of HDAC2 and DNMT1 blocking the 5'CpG island methylation of the tumour suppressor genes resulting to transcriptional activation by upregulation of their mRNA. There was histone hyperacetylation which opens chromatin structure in which the DNA is more loosely wrapped around the histones making it more receptive to interaction with transcription factors.Overexpression of the TSGs combined with the MT depolymerizing action of VRL inhibited metabolic activity and DNA synthesis of tumor cells according to MTT and BrdU.Immunological analysis exhibited ADCC.There was induction of PCD in IDC cells according to TUNEL and TEM.A large number of tumor cells formed apoptotic bodies which were phagocytosed by adjacent tumor cells leading to a bystander killing effect.Concluding,this therapeutic approach with immunovinorelbine (IVRL) may revolutionize IDC treatment adding significantly to the current clinical armamentarium. No significant financial relationships to disclose.

Immunochemoconjugate of anti-DNMT1/HDAC2 bispecific F(ab)2-bsAb linked with cleavable disulfide to vinorelbine induces apoptosis in chemoresistant infiltrating ductal carcinoma (IDC) characterised by HDAC2 overexpression and 5'CpG island hypermethylation of the FHIT, RAR-β2, BRCA-1, APC, p16(CDKN2A), RASSF1A, CDH1(E-cadherin), 14–3-3-σ (stratifin), HIC1 and MDG1 tumor suppressor genes

2088 One of the reasons for chemoresistance to IDC is deacetylation and DNA methylation which causes silencing of tumor suppressor genes(TSGs).DNMT1 interacts with HDAC2 to repress transcription of TSGs. IDC cells obtained by surgical excision from a patient were analysed by IHC,PCR-based LOH, ChIP, MSP,RT-PCR and NB.There was loss of FHIT expression,LOH at FHIT and 5' CpG island methylation of the FHIT .There was transcriptional silencing of the following TSGs: BRCA1,RAR-β2,APC,p16 (CDKN2A), RASSF1A,CDH1(E-cadherin),14–3-3-σ (stratifin),HIC1 and MDG1.There was overexpression of DNMT1 and HDAC2 suggesting a link between histone deacetylation,cytosine methylation,local chromatin condensation and subsequent transcriptional repression.This chemoresistant IDC was defined as CIMP+.We treated IDC cells with anti-DNMT1/HDAC2 bispecific F(ab)2-bsAb linked with cleavable disulfide to VRL . Post-treatment, there was inhibition of HDAC2 and DNMT1 blocking the 5'CpG island methylation of the tumour suppressor genes resulting to transcriptional activation by upregulation of their mRNA. There was histone hyperacetylation which opens chromatin structure in which the DNA is more loosely wrapped around the histones making it more receptive to interaction with transcription factors.Overexpression of the TSGs combined with the MT depolymerizing action of VRL inhibited metabolic activity and DNA synthesis of tumor cells according to MTT and BrdU.Immunological analysis exhibited ADCC.There was induction of PCD in IDC cells according to TUNEL and TEM.A large number of tumor cells formed apoptotic bodies which were phagocytosed by adjacent tumor cells leading to a bystander killing effect.Concluding,this therapeutic approach with immunovinorelbine (IVRL) may revolutionize IDC treatment adding significantly to the current clinical armamentarium. No significant financial relationships to disclose.

Effect of murine liver cell proliferation on herpes viral behavior: implications for oncolytic viral therapy.

Replication-competent herpes simplex oncolytic viruses are promising anticancer agents that partly target increased DNA synthesis in tumor cells. Investigators have proposed that these DNA viruses may be combined with liver resection to enhance killing of liver malignancies. Whether or not the cellular alterations associated with hepatic regeneration affect the efficacy and toxicity of these promising anticancer agents is unknown. This study examined the behavior of two oncolytic viruses, NV1020 and G207, during liver regeneration. When delivered during the peak of liver regeneration, replication and appearance of both G207 and NV1020 in hepatic tissue are enhanced as demonstrated by histochemical staining for the marker gene lac Z, immunohistochemical staining, and quantitative polymerase chain reaction. This increased appearance of virus in liver tissue correlates with increases in cellular ribonucleotide reductase activity and DNA synthesis and is also associated with increased viral binding. However, increased viral presence is transient, and viral detection declines to baseline within 7 days. When these viruses were delivered to animals even as early as 7 days after hepatectomy, there proved to be no measurable viral replication in any organ and no increased morbidity or mortality. In conclusion, the early stages of hepatic regeneration after resection provide an environment suitable for viral replication. Administration of replication-competent herpes simplex virus during the peak of hepatocyte regeneration (24-48 hours) permits viral productivity in tissue that otherwise does not support viral growth. The increase in hepatotoxicity after hepatectomy is short-lived and can be predicted by peak hepatocyte DNA synthesis.

A garlic lectin exerted an antitumor activity and induced apoptosis in human tumor cells

Cytotoxic effects of a lectin prepared from garlic ( Allium sativum-L) bulbs on human tumor cells were studied. The lectin strongly reduced the growth and DNA synthesis of human tumor cells in a time- and a dose-dependent manner. By contrast, a soybean lectin showed only a weak inhibitory effect on growth and DNA synthesis of tumor cells. Furthermore, the garlic lectin induced apoptosis in the cells at a low concentration. The antitumor activity of garlic lectin may provide a rational basis for its effectiveness observed in clinical applications.

Effect of iscador, a drug from mistletoe (Viscum album), on DNA synthesis in tumor cells and the functional activity of normal phagocytic cells

Effect of iscador, a drug from mistletoe (Viscum album), on DNA synthesis in tumor cells and the functional activity of normal phagocytic cells

Hormonal regulation of proliferation of granulosa and Leydig cell lines derived from gonadal tumors of transgenic mice expressing the inhibin-α subunit promoter/simian virus 40 T-antigen fusion gene

We have produced a transgenic (TG) mouse model expressing the Simian Virus 40 T-antigen (Tag) gene, driven by a 6-kb fragment of the mouse inhibin-α subunit promoter (inh-α). The mice develop gonadal tumors with 100% penetrance by the age of 5–8 months, of granulosa cell origin in the ovary, and of Leydig cell origin in the testis. In the present study, we characterized the hormonal regulation of proliferation of two immortalized cell lines, BLT-1, originating from a Leydig cell tumor, and NT-1, originating from a granulosa cell tumor. [ 3H]-thymidine incorporation in both types of cells was stimulated by activin (≥10–30 μg/l), while inhibin had no effect. Transforming growth factor (TGF)-β, at ≥0.01 μg/l, stimulated proliferation of the granulosa tumor cells, but no effect was found on the Leydig tumor cells. Progesterone inhibited the proliferation of both cell lines, although the granulosa tumor cells were clearly less sensitive than the Leydig cells to this effect (≥3 μmol/l vs. >10 nmol/l, respectively). hCG had no effect on the Leydig tumor cell DNA synthesis whereas at high concentration (100 μg/l) it stimulated that of the granulosa cells. We also investigated in BLT-1 and NT-1 cells whether the proliferative changes were related to concomitant changes in Tag expression. In BLT-1 cells, this was stimulated by activin, progesterone and hCG, even though the latter substance did not affect cell proliferation. In contrast, TGF-β inhibited Tag expression. In NT-1 cells, the expression of Tag was stimulated by activin, while hCG had no effect. In contrast, it was reduced by progesterone, inhibin and TGF-β. In conclusion, our results indicate that the granulosa and Leydig tumor cells, despite similar mechanism of immortalization, respond differently to several mitotic stimuli. The responses in the level of Tag expression in these cells did not always correlate with the changes observed in cell proliferation, indicating the independence of these two phenomena.

A chelating agent possessing cytotoxicity and antimicrobial activity: 7-Morpholinomethyl-8-hydroxyquinoline

7-Morpholinomethyl-8-hydroxyquinoline (MO-8HQ), which like 8-hydroxyquinoline (8HQ) readily forms a chelate, was synthesized and found to possess cytotoxicity and antimicrobial activity. Both 8HQ and MO-8HQ were cytotoxic to human carcinoma cell lines at micromolar concentrations. MO-8HQ also inhibited DNA synthesis of tumor cells at micromolar concentrations, suggesting that MO-8HQ might chelate metals necessary for the enzymatic catalysis of DNA biosynthesis. MO-8HQ was more active against Gram positive bacteria than Gram negative bacteria and its potency correlated with iron chelation. An “unsaturated” chelate with a MO-8HQ to Fe ratio of 2:1 exhibited greater antibacterial activity than MO-8HQ alone. Among the organisms tested, Micrococcus flavus was most susceptible with a MIC of 3.9 μg ml . MO-8HQ also exhibited anti-fungal activity at 7–15 μg ml . MO-8HQ:Fe chelate markedly increased the susceptibility of Escherichia coli to deoxycholate. Addition of Ca 2+ or Mg 2+ reversed the sensitivity of bacteria to deoxycholate as well as to rifampicin. It is suggested that MO-8HQ exerts its biological activity as a membrane-active agent through metal ion chelation.

Comparative analysis of radiometric and autoradiographic methods in the investigation of DNA synthetic activity of tumor cells in vitro

DNA synthesis in cell cultures of Ehrlich ascites carcinoma (EAC) was studied by means of autoradiography and liquid scintillation radiometry. The following parameters were compared for estimation of DNA synthesis: a) total radioactivity of incorporated [H-3]thymidine registered by a liquid scintillation counter, b) labeling index (LI) reflecting the relative number of cells which synthesize DNA, and c) mean grain count (MGC) indicating average intensity of DNA synthesis in the S-phase of the cell cycle. Kinetics of total radioactivity of incorporated [H-3]thymidine was determined to correlate better with LI. Changes in DNA synthesis in vitro were shown to be monitored with a higher level of significance by the radiometric technique, but autoradiography was more sensitive in some cases. Priority and informative value of the two methods were discussed. Autoradiography and liquid scintillation radiometry were concluded to be mutually complementary for investigation of DNA synthesis in tumor cells in vitro. Combination of the two methods was recommended for investigation of influence of cell proliferation inhibitors on DNA synthesis.

Effect of ultrasound on DNA synthesis in tumor cells

The objective of this study was to consider the influence of continuous-wave (CW) ultrasound (800 kHz) on DNA synthesis in Ehrlich ascitic tumor cells in vitro. The 10-min irradiation was carried out in rotating polyethylene tubes (5 rpm). Incorporation of 3H-thymidine was employed to detect DNA synthesis. Replication and unscheduled (repair) DNA synthesis were distinguished by means of hydroxyurea treatment before insonation. It was established that the use of 0.5 W/cm 2 and 1.0 W/cm 2 ( I SA ) CW ultrasound interfered with DNA synthesis showing up as inhibition. The inhibition became most noticeable when starting insonation in a sample cooled to 5°C. During insonation, the sample temperature was allowed to increase to the ambient (water bath) temperature of 37°C. However, at an ultrasound intensity (SA) of 0.1 W/cm 2 such inhibition was not proven, but a moderate stimulation of DNA synthesis was demonstrated. Changes in DNA synthesis after insonation were found to be transient because of a lack of stimulatory and/or inhibitory ultrasound effects after 1-h incubation at 37°C. In our experimental conditions no effects on DNA synthesis due to the effects of the insonated medium on the cells were discernible. No statistically significant changes in unscheduled DNA synthesis were observed. Possible action mechanisms are discussed.

Immunocytochemical demonstration of melanotransferrin (p97) in thyroid tumors of follicular cell origin

Using the PAP method on paraffin sections, we investigated the distribution pattern of melanotransferrin (p97) in follicular adenomas (20 cases), atypical adenomas (4 cases), papillary (10 cases) and follicular carcinomas (25 cases) of the thyroid; frozen sections collected from 3 follicular adenomas and 2 follicular carcinomas were also tested. Our results document positivity for p97 in atypical adenomas, papillary and follicular carcinomas, whereas no reactivity is encountered in follicular adenomas and normal portions of thyroid. We suggest that the production of p97 by neoplastic cells of atypical adenomas and differentiated thyroid carcinomas could result in a greater availability of iron for tumor cell DNA synthesis.

Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide.

Macrophage-derived nitric oxide (NO) is cytostatic to tumor cells and microbial pathogens. We tested whether one molecular target for the cytostatic action of NO may be ribonucleotide reductase (RR), a rate-limiting enzyme in DNA synthesis. In a concentration-dependent manner, NO gas and lysates of activated macrophages that generated comparable amounts of NO led to the same degree of inhibition of partially purified RR from L1210 mouse lymphoma cells. Lysates from nonactivated macrophages, which do not produce NO, were noninhibitory. With lysates from activated macrophages, RR was protected by omitting L-arginine or by adding the NO synthase inhibitors diphenyleneiodonium, N omega-methyl-L-arginine, or N omega-amino-L-arginine. L-Arginine, but not D-arginine, abolished the protective effect of N omega-amino-L-arginine. The prototypic pharmacologic inhibitor of RR is hydroxyurea. Its structural resemblance to N omega-hydroxy-L-arginine, a reaction intermediate of NO synthase, prompted us to test if hydroxyurea can generate NO. In the presence of H2O2 and CuSO4, hydroxyurea produced NO2-/NO3-, aerobic reaction products of NO. Addition of morpholine blocked NO2-/NO3- generation from hydroxyurea and led to formation of nitrosomorpholine, as detected by gas chromatography/mass spectrometry. Thus, hydroxyurea can produce an NO-like, nitrosating rectant. L1210 cell DNA synthesis was inhibited completely by activated macrophages or by hydroxyurea, and was partially restored to the same degree in both settings by providing deoxyribonucleosides to bypass the block in RR. Thus, both NO gas and NO generated by activated macrophage lysates inhibit tumor cell RR. The RR inhibitor hydroxyurea can also generate an NO-like species. Similar, partial restoration of tumor cell DNA synthesis by deoxyribonucleosides in the presence of activated macrophages or hydroxyurea suggests that cytostasis by activated macrophages and by hydroxyurea has comparable mechanisms, including, but probably not limited to, inhibition of RR.