Incorporation Of Nucleic Acid Precursors Research Articles

Prediction of Cancer Drug Resistance and Implications for Personalized Medicine.

Drug resistance still impedes successful cancer chemotherapy. A major goal of early concepts in individualized therapy was to develop in vitro tests to predict tumors’ drug responsiveness. We have developed an in vitro short-term test based on nucleic acid precursor incorporation to determine clinical drug resistance. This test detects inherent and acquired resistance in vitro and transplantable syngeneic and xenografted tumors in vivo. In several clinical trials, clinical resistance was predictable with more than 90% accuracy, while drug sensitivity was detected with less accuracy (~60%). Remarkably, clinical cross-resistance to numerous drugs (multidrug resistance, broad spectrum resistance) was detectable by a single compound, doxorubicin, due to its multifactorial modes of action. The results of this predictive test were in good agreement with predictive assays of other authors. As no predictive test has been established as yet for clinical diagnostics, the identification of sensitive drugs may not reach sufficiently high reliability for clinical routine. A meta-analysis of the literature published during the past four decades considering test results of more than 15,000 tumor patients unambiguously demonstrated that, in the majority of studies, resistance was correctly predicted with an accuracy between 80 and 100%, while drug sensitivity could only be predicted with an accuracy of 50–80%. This synopsis of the published literature impressively illustrates that prediction of drug resistance could be validated. The determination of drug resistance was reliable independent of tumor type, test assay, and drug used in these in vitro tests. By contrast, chemosensitivity could not be predicted with high reliability. Therefore, we propose a rethinking of the “chemosensitivity” concept. Instead, predictive in vitro tests may reliably identify drug-resistant tumors. The clinical consequence imply to subject resistant tumors not to chemotherapy, but to other new treatment options, such as antibody therapy, adoptive immune therapy, hyperthermia, gene therapy, etc. The high accuracy to predict resistant tumors may be exploited to develop new strategies for individualized cancer therapy. This new concept bears the potential of a revival of predictive tests for personalized medicine.

Vascular thickness and cytostatic-resistant bronchial carcinoma

A new way of treatment of malignant tumours was suggested by blocking angiogenesis and thus creating subpopulations of cells which, due to their decreased oxygen content, react differently to treatment. The relationship between angiogenesis (vessel density) and resistance to cytostatic drugs was investigated. Results were analysed retrospectively for 83 untreated patients (73 men, 10 women; mean age 60 [37-75] years) with non-small cell bronchial carcinoma. Vessel density and resistance proteins were determined immunohistochemically. Radioactive incorporation of nucleic acid precursors with and without doxorubicin was measured with the in vitro short-term test. There were significant differences between vessel density and various resistance proteins. With low vessel density (less than mean value) glutathione-S-transferase pi (P < 0.01), thymidylate synthase (P < 0.05) and metallothioneine (P < 0.05) were significantly increased. P-glycoprotein showed a corresponding relationship, but it was not statistically significant, while there was no relationship with topoisomerase II. Bronchial carcinoma with low vessel density was more frequently resistant in vitro to doxorubicin than carcinomas with higher vessel density (P < 0.05). Non-small cell bronchial carcinoma with low vessel density more frequently expresses resistance proteins and is more commonly resistant to doxorubicin. Blocking angiogenesis to improve the results of treatment is therefore problematic.

Characterization of a streptococcal antitumor glycoprotein (SAGP)

An acidic antitumor glycoprotein (SAGP) was purified from a crude extract of Streptococcus pyogenes, Su strain. Intraperitoneal injection with SAGP (20 mg protein/kg/day for 4 consecutive days) prolonged the life span of mice inoculated i.p. with Ehrlich ascite carcinoma cells and methylcholanthrene-induced fibrosarcoma cells (Meth A) up to 244% and 169% of that of the control mice, respectively. These in vivo antitumor effects were reduced in immunosuppressed mice. The effector spleen cells from the Meth A-inoculated and SAGP-injected mice showed a considerable cytostatic activity on Meth A cells in vitro, and immunosuppression studies suggested that carrageenan-sensitive and/or asialo-GM1 positive spleen cells are responsible for the in vivo antitumor effect of SAGP. SAGP inhibited the cell growth of cultured cell lines including transformed hamster embryonic lung cells, murine leukemia L1210, Meth A and human promyelocytic leukemia HL60 cells. The IC 50s for the cell growth of these cells were all below 0.1 μg protein/ml. SAGP inhibited the incorporation of nucleic acid precursors into Meth A cells. It seems that sulfhydryl groups of the SAGP molecule are essential for the expression of the antitumor action of SAGP. The cell growth-inhibitory activity of SAGP was diminished in Meth A cells preincubated with pertussis toxin (IAP), whereas it was augmented in the cells preincubated with cholera toxin (CTX), suggesting the involvement of toxin-sensitive GTP (G)-proteins in the SAGP-action. IAP and CTX-catalyzed ADP ribosylation assays confirmed that SAGP augmented the activity of IAP-sensitive G-protein. In addition, this augmentation was detected neither in Meth A cells incubated with heat-inactivated SAGP nor in SAGP-insensitive L929 cells. SAGP induced apoptosis in Meth A and HL60 cells as assessed by DNA fragmentation. A single dose injection of SAGP (100mg protein/kg, i.v., s.c., or i.p.) into mice produced no tixic signs except occasional pain responses observed for one week after the injection. Thus, SAGP is a low toxic substance that shoes in vivo antitumor activity by modulating immune responses of the host, and also exhibits in vitro cell-growth inhibition through IAP-sensitive G-protein.

Streptococcal glycoprotein-induced tumour cell growth inhibition involves the modulation of a pertussis toxin-sensitive G protein.

We studied the mechanism of anti-tumour action of sulphydryl glycoprotein (SAGP) purified from an extract of Streptococcus pyogenes in vitro. SAGP rapidly inhibited the incorporation of nucleic acid precursors into murine fibrosarcoma (Meth A) cells before it inhibited the cell growth. SAGP-induced cell growth inhibition was diminished by incubating the cells with pertussis toxin (IAP), whereas the SAGP activity was augmented by incubating the cells with cholera toxin (CTX). Meth A cells exposed to SAGP underwent an increase in labelling of the alpha-subunit of an inhibitory guanine nucleotide-binding (Gi) protein in a subsequent IAP-catalysed [32P]ADP ribosylation of the cell membrane fraction. Gi alpha labelling was not increased either in the membrane from the Meth A cells exposed to heat-inactivated SAGP or in the membrane from L929 cells exposed to SAGP, in which growth was also unaffected. By contrast, SAGP caused no alteration in labelling the alpha-subunit of stimulatory guanine nucleotide-binding (Gs) protein in a subsequent CTX-catalysed ADP ribosylation of membrane fractions of Meth A and L929 cells. The amount of intracellular cAMP was decreased slightly in Meth A cells incubated with SAGP. Although the precise roles of Gs protein and adenylate cyclase in the cell growth inhibition induced by SAGP are not clear, these findings suggested that the modulation of Gi protein is involved in such SAGP-induced cellular events as the inhibition of nucleic acid synthesis and cell growth inhibition.

Metabolism of carbovir, a potent inhibitor of human immunodeficiency virus type 1, and its effects on cellular metabolism.

Carbovir (CBV) [the (--)-enantiomer of the carbocyclic analog of 2',3'-dideoxy-2',3'-didehydroguanosine] is a potent inhibitor of human immunodeficiency virus type 1 (HIV) replication in vitro. We have characterized the metabolism of CBV and its effect on cellular metabolism in an effort to better understand its mechanism of action. CBV was primarily metabolized to the 5'-triphosphate of CBV (CBV-TP) to concentrations sufficient to inhibit HIV reverse transcriptase. Infection of CEM cells with HIV did not affect the metabolism of CBV. In CEM cells, there was no evidence of the degradation of CBV by purine nucleoside phosphorylase. The half-life of CBV-TP in CEM cells was 2.5 h, similar to that of the 5'-triphosphate of zidovudine (AZT). However, unlike the levels of the 5'-triphosphate of AZT, CBV-TP levels declined without evidence of a plateau. CBV did not affect the metabolism of AZT, and AZT did not affect the metabolism of CBV. A small amount of CBV was incorporated into DNA in intact CEM cells, and this incorporation was increased by incubation with mycophenolic acid, an inhibitor of IMP dehydrogenase. CBV specifically inhibited the incorporation of nucleic acid precursors into DNA but had no effect on the incorporation of radiolabeled precursors into RNA or protein. CBV did not decrease the level of TTP, dGTP, dCTP, or dATP. These results suggested that the cytotoxicity of CBV was due to the inhibition of DNA synthesis. Further studies are necessary to identify the target(s) responsible for growth inhibition.

In vitro activity of 4′ -iodo-4′ -deoxydoxorubicin on human colo-rectal cancer as measured by a short-term antimetabolic assay

A short-term antimetabolic assay based upon the inhibition of incorporation of nucleic acid precursors was used to compare the cytotoxicity of a new halogenated anthracycline, 4'-iodo-4'-deoxydoxorubicin (IDX), with that of its parent compound doxorubicin (DX) on human colo-rectal carcinoma specimens. IDX showed a marked dose-dependent effect, with frequencies of activity consistently greater than those of DX at all concentrations. The minimal dose required to induce a significant antimetabolic effect for IDX was 1/10 that for DX.

In vitro Sensitivity Test of Breast Cancer Cells to Hormonal Agents in a Radionucleotide‐incorporation Assay

Breast cancer cell lines (MCF‐7, T47D, BT‐20 and STT‐11) and fresh cells from malignant effusions of eight breast cancer patients were examined for their in vitro sensitivity to 17β‐estradiol (E2), tamoxifen and toremifene in a miniaturized, improved nucleic acid precursor incorporation assay (MINI assay). Seven of the eight patients received either tamoxifen or toremifene following a MINI assay and the correlation was examined between in vitro sensitivity and clinical responses to the hormonal agents. In cell lines, E2 stimulated thymidine incorporation by estrogen receptor (ER)‐rich cells, MCF‐7 and T47D, but not by ER‐poor cells, BT‐20 and STT‐11. Tamoxifen induced both ER‐mediated and ‐unmediated effects in ER‐rich cells. The latter effect was also observed in ER‐poor cells. Toremifene had less ER‐unmediated effect in all of the cells tested than tamoxifen did. The ER‐mediated effect of toremifene was weaker than that of tamoxifen in cell lines but was equipotent to tamoxifen in fresh cells. E2 affected thymidine incorporation by cells withdrawn from patients who showed a partial response to the anti‐estrogens. No clear correlation was demonstrated between in vitro sensitivity to anti‐estrogens of fresh cells and clinical response to these agents. The present results suggest that 1) the MINI assay is a useful system to investigate hormonal effects on breast cancer cell lines; 2) clinical responses to anti‐estrogens are not predicted by in vitro response to the agents but might be predicted by the in vitro response to E2; and 3) toremifene has a smaller non‐specific effect on breast cancer cells than tamoxifen and is equipotent to tamoxifen in the ER‐mediated effect in vitro.

In vitro chemosensitivity testing in the treatment of ovarian carcinoma.

In vitro chemosensitivity to cisplatinum, adriamycin, cyclophosphamide, and 5-fluorouracil was investigated in 58 cases of ovarian carcinoma using Volm's short-term test. These in vitro results were retrospectively correlated with the relapse-free interval. Operative treatment in all patients (FIGO stage I (5), III (43), IV (10)) comprised maximum debulking procedure including hysterectomy, adnexectomy, omentectomy, pelvic and in most cases additionally paraaortic lymphadenectomy. Subsequently, all patients were treated with the cisplatinum-epirubicin-cyclophosphamide regimen. 33/58 tumors (66%) were sensitive in vitro (inhibition of nucleic acid precursor incorporation of more than 45% as compared to untreated controls). The median relapse-free interval of patients with sensitive tumors was significantly longer than that of those with resistant carcinomas (30.3 versus 22.6 months, respectively; P less than 0.05). Histopathological evaluation showed the majority of serous cystadenocarcinomas to be sensitive (26/33 = 79%, P less than 0.05).

Mechanism of action of interferon-alpha 2 in hairy cells and lymphoblastoid cell lines: the significance of type I interferon receptors and RNA synthesis

Lymphoblastoid cells were subjected to Western Blot analysis for detection of Interferon-alpha-binding proteins. JOK-1 cells--a human hairy cell leukemia line--revealed three proteins with apparent molecular weights of 120, 100 and 32 kD, respectively. Down-regulation of the receptor was observed. A differential binding pattern of two proteins (100 and 85 kD) was observed in T-CLL, whereas no signal detection was achieved in B-CLL. Mononuclear cells from 6 patients with hairy cell leukemia and 6 patients with CLL were found to differ significantly in terms of nucleic acid precursor incorporation.

Chemosensitivity testing of small biopsy specimens

AbstractChemosensitivity testing has usually been limited to tumor specimens of 2–3 g or more. We evaluated the miniaturized, improved nucleic acid precursor incorporation assay (MINI‐assay) in 100 small biopsy specimens of 1 g or less. Tumor types included malignant melanomas; sarcomas; and breast, colon, lung, and ovarian carcinomas. Overall, 74/100 were evaluable. The evaluability rate was related to size: 41/48 (85%) 0.7–1.0‐g specimens were evaluable, as opposed to 22/35 (64%) 0.4–0.6‐g and 11/17 (64%) 0.1–0.3‐g specimens. Evaluability was not correlated with tumor types or whether the tumor was from a primary or metastatic site, but was related to yield of viable tumor cells. No viable tumor cells were obtained in 17 specimens, and thus no assay was performed. When 105 cells per well were plated, 49/54 (91%) assays were evaluable, but only 25/29 (86%) were successful when fewer cells were used. Four or more anticancer drugs could be tested in 69/74 (95%) of the evaluable assays. Yielding high evaluability rates and requiring fewer viable cells, the MINI‐assay has allowed us to perform chemosensitivity tests on previously unusable biopsy specimens.

Reliability of an in vitro short-term assay to predict the drug sensitivity of human breast cancer.

The feasibility and reliability of an in vitro assay that evaluates drug interference on nucleic acid precursor incorporation were investigated on 135 previously untreated locally advanced breast cancers. The assay, which was carried out on tumor fragments incubated for 3 hours with drugs, proved to be feasible on a sufficiently high percentage of biopsy specimens (70%) for routine clinical use. In vitro drug activity evaluated with this assay appeared to reproduce the clinical patterns of sensitivity of the tumor type as well as of the individual tumors. In fact, in vitro response rates to conventional agents resembled the clinical response rates reported for the same agents used in monochemotherapy. From a retrospective--correlative study carried out on 41 patients treated in vitro and in vivo with the same drugs (Adriamycin [doxorubicin] and vincristine), in vitro effect of Adriamycin on 3H-uridine incorporation appeared significantly correlated with clinical response (overall agreement, 78%; P = 0.0032) with specific agreements of sensitivity and resistance of 75% and 81%, respectively.

Monoclonal Antibodies to Trypanosoma cruzi Inhibit Motility and Nucleic Acid Synthesis of Culture Forms

Monoclonal antibodies were raised against the surface of epimastigotes and metacylic trypomastigotes of Trypanosoma cruzi, as shown by electron microscopy, agglutination, and immunofluorescence. The antibodies were stage specific but not strain specific. A deleterious effect of the antibodies on T. cruzi culture forms was shown by the drastic reduction of parasite motility and incorporation of nucleic acid precursors. Some fraction of the parasite population, however, was viable and replicated and infected mouse macrophages in culture. The antibodies were found to also mediate complement-induced lysis of culture forms of T. cruzi.

The effect of manganese limitation on DNA precursor biosynthesis during nucleotide fermentation with Brevibacterium ammoniagenes and micrococcus luteus

The induction of unbalanced growth by manganese starvation has been confirmed for Brevibacterium ammoniagenes ATCC 6872 and has also been investigated with Micrococcus luteus ATCC 15932. In both bacteria the interruption of DNA production found in cells lacking manganese is reversible. DNA production but not RNA, protein or peptidoglycan synthesis can be selectively restored by the addition of Mn2+. After manganese reactivation, starved cells showed enhanced incorporation of nucleic acid precursors into the DNA fraction compared with the manganese-supplied cells. This recovery of DNA production in M. luteus was severely inhibited by hydroxyurea (HU). Recovery was not inhibited by chloramphenicol or rifampicin in the case of simultaneous addition of Mn2+. HU is known to be a specific inhibitor of ribonucleotide reduction, supplying monomeric precursors for DNA synthesis. Impairment of DNA precursor biosynthesis due to inactivation of a manganese-activated ribonucleotide reductase is a possible explanation for the reversible interruption of DNA production observed in both coryneform bacteria under conditions of manganese deficiency.

Glucocorticoid receptors and their functions in lymphocytes

Most, if not all. the effects of glucocorticoid hormones on lymphoid tissue are thought to be mediated by an initial step of interaction of the steroid with specific cytoplasmic receptors. We have presented results, obtained using whole cell suspensions of mouse thymocytes. showing however that the number of receptors does not accurately reflect the steroid sensitivity of the target cell: both inhibitory effects of steroid on nucleic acid precursor incorporation and steroid-induced cell lysis are not obligatorily related to receptor levels but more likely to cell differentiation. We have therefore also investigated the mechanism of the immunosuppressive effects of steroids and the role of various factors which could be involved in the proliferation or lysis of mouse thymocytes. Dexamethasone and 25-OH cholesterol are the most potent agents, capable of inhibiting cholesterol biosynthesis in non stimulated thymocytes. Although the effect of dexamethasone. which is blocked by actinomycin D. appears to be mediated through receptor occupancy, the action of 25-OH cholesterol is likely to be due to an inhibition of HMG COA reductase activity. Mitogen induced lymphoblastic transformation of thymocytes is blocked by low concentration of dexamethasone but also by sex steroids at higher concentrations. These compounds, which exert immunosuppressive effects in vivo, inhibit concanavalin A induced transformation at concentrations above 10 −5 M, at which concentration they also elicit a rapid and transient decrease of uridine uptake in non stimulated thymocytes. In addition, calcium, which has been considered to play an important role in the mechanism of steroid-induced lysis, appears highly toxic by itself.

Differences in incorporation of nucleic acid bases and nucleosides by various Mycoplasma and Acholeplasma species.

Eight species representative of the serological diversity of the Mycoplasmatales were tested for their ability to incorporate radiolabeled nucleic acid precursors into acid-insoluble material. Cultures in complex growth medium were centrifuged and resuspended in minimal essential medium (Eagle). For Acholeplasma laidlawii, labeling occurred mainly during the first 4 h of incubation, with substrate saturation at 20 micron. All organisms tested incorporated uracil, adenine, and guanine; none incorporated cytosine. Thymine was incorporated only by bovine group 7, Mycoplasma putrefaciens, and Mycoplasma pneumoniae (strain 3546), but deoxynucleosides enhanced thymine incorporation in A. laidlawii, Mycoplasma gallisepticum, M. pneumoniae (strain AP-164), and Mycoplasma hyorhinis. Nucleoside incorporation (adenosine, guanosine, uridine, cytidine, and thymidine) was not observed for the arginine-utilizing species, Mycoplasma hominis and Mycoplasma arginini, whereas all other organisms tested incorporated nucleosides. The incorporation pattern provides additional metabolic evidence to support the biochemical and antigenic diversity of these organisms. The recognition of differences in incorporation of nucleic acid precursors is important not only to the specific labeling of these organisms, but also to the study of metabolism and transport.

Autoradiographic study of nucleic acid precursor incorporation in nerve cells of the mouse brain

Following intravenous administration of five tritiated nucleic acid bases and five nucleosides in mice the intensity of labelling of the cerebral cortex cells and the liver cells show the following pattern. Cytidine and uridine cause intense labelling; uracil, adenine, adenosine, guanosine, guanine, thymine and cytosine only a weak labelling. The choroid plexus is labelled intensely by cytidine and uridine, moderately by guanosine and adenosine, weakly by other substances. In vitro experiments and intracerebral injection with uridine, uracil, adenine and adenosine give the same labelling pattern as intravenous injection. Inhalation of CO 2 does not alter the labelling of nerve cells apart from that due to the uridine group, where a weaker labelling of the nerve cells is observed. It is concluded that, in the case of the nucleic acid precursors used here, metabolic specificities are of greater significance for the labelling pattern of the nerve cells than the transport specificities of the blood-brain barrier.

Mitotic activity and nucleic acid precursor incorporation in denervated and innervated limb stumps of axolotl larvae.

Mitotic activity and DNA and RNA precursor incorporation were compared in innervated regenerating limbs and in denervated, non-regenerating limbs on days 8 and 9 post-amputation. Innervated limbs had well-developed cone stage blastemas which showed high cellular mitotic indices and H3-thymidine labeling indices of 0.40-0.50 and H3-uridine labeling indices of 0.50-0.75. In contrast, denervated limbs showed dedifferentiated cells distally under thickened wound epithelia, but essentially no mitotic activity and no blastema formation. These dedifferentiated cells showed lower levels of H3-thymidine (0.10 index) and H3-uridine (0.50) incorporation than regenerating limbs. Labeling indices of wound epithelia are also compared.

Altered incorporation of nucleic acid precursors by mycoplasma-infected mammalian cells in culture

Established cultures of 3T3 cells and HeLa S-3 cells infected with mycoplasma exhibited various alterations in their labeling patterns when exposed to tritiated nucleosides. The alterations were manifested by diffuse cytoplasmic labeling and the absence of continuous incorporation of the precursors into acid insoluble fractions. This anomaly appeared only when the growth medium was supplemented with serum, which contains essential nutrients for many species of mycoplasma. Our experiments showed that the alterations in incorporation could be attributed to depletion of the exogenous thymidine or uridine, due to their enzymatic degradation by the microorganisms into the free base and sugar moieties. Other experiments revealed that uracil was utilized by the mycoplasma. The rate of degradation of the nucleosides was quantitated by the paper chromatographic separation of the breakdown products. Because of the relative ease and high sensitivity of this method, its routine application is suggested as a means of revealing hidden mycoplasmal contamination not readily detectable by standard microbiological techniques.

Stimulation of incorporation of nucleic acid precursors into HeLa cells caused by proflavine.

1. The effect of proflavine and other acridines on the incorporation of precursors into the nucleic acids of HeLa cells was examined. 2. Relatively low concentrations (50mum) of proflavine completely inhibited incorporation of precursors into DNA, but allowed a small extent of incorporation into RNA. 3. Acridine-resistant incorporation into RNA was unaffected by actinomycin D at 2mug./ml. and persisted even at high concentrations (500mum) of many acridines. 4. A few combinations of acridine and precursor, notably 250mum-proflavine and [(14)C]adenine, caused a stimulation of incorporation. 5. The proflavine-stimulated incorporation was into alkali-stable di- and tri-nucleotides. 6. It was concluded that the effect was due to the preferential inhibition of degradation of a fraction of RNA that normally turned over, thus allowing small radioactive oligonucleotides to accumulate in the cells.

The inner membrane of the rat liver mitochondrion as the site of incorporation of radioactively labeled precursors into nucleic acid and protein in vitro

The experiments reported in this study confirm the role of the inner membrane of rat liver mitochondria in the incorporation of radioactive amino acids into protein in vitro and extend that role to include the incorporation of nucleic acid precursors. Purified inner membrane preparations, prepared by a digitonin method, were shown to be capable of effecting the energy-dependent incorporation of 14C-leucine into membrane protein. The process was inhibited by puromycin and chloramphenicol but not by cycloheximide. Actinomycin D also inhibited the reaction. The incorporation of 3H-ATP into mitochondrial RNA and 3H-dATP into DNA by these membranes in vitro was found to be dependent on Mg 2+ and partially dependent on the presence of all four ribo- or deoxyribonucleoside triphosphates. The incorporation of both of these precursors was inhibited by actinomycin D. Inner membranes, prepared from mitochondria by osmotic lysis or sonic vibration, were inactive. Furthermore, active inner membrane preparations prepared by the digitonin method and subsequently exposed to water or sonic treatment were also inactivated. These results suggest that the inner membrane of rat liver mitochondria is capable of incorporating precursors of both nucleic acids and proteins and might carry out the reactions leading from mitochondrial DNA → RNA → membrane protein and that a minimal structural organization might be necessary for the process.