pentostatin and Colonic-Neoplasms

The combination of 2'-deoxyadenosine and 2'-deoxycoformycin is toxic for the human colon carcinoma cell line LoVo. In this study we investigated the mode of action of the two compounds and have found that they promote apoptosis. The examination by fluorescence microscopy of the cells treated with the combination revealed the characteristic morphology associated with apoptosis, such as chromatin condensation and nuclear fragmentation. The occurrence of apoptosis was also confirmed by the release of cytochrome c and the proteolytic processing of procaspase-3 in cells subjected to the treatment. To exert its triggering action on the apoptotic process, 2'-deoxyadenosine enters the cells through an equilibrative nitrobenzyl-thioinosine-insensitive carrier, and must be phosphorylated by intracellular kinases. Indeed, in the present work we demonstrate by analysis of the intracellular metabolic derivatives of 2'-deoxyadenosine that, as suggested by our previous findings, in the incubation performed with 2'-deoxyadenosine and 2'-deoxycoformycin, an appreciable amount of dATP was formed. Conversely, when also an inhibitor of adenosine kinase was added to the incubation mixture, dATP was not formed, and the toxic and apoptotic effect of the combination was completely reverted.

Topics: Antineoplastic Agents; Apoptosis; Caspases; Cell Adhesion; Cell Count; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Deoxyadenosines; Enzyme Precursors; Flow Cytometry; Humans; Microscopy, Fluorescence; Pentostatin; Thioinosine

In LoVo cells, phosphorolytic activity acting on deoxyadenosine plays a major role in the resistance to the cytotoxic effect of the combination of deoxynucleoside with deoxycoformycin. In fact, the observed dependence of toxicity on cell density appears to be related to the metabolic conversion of deoxyadenosine into adenine. The phosphorylation of the deoxynucleoside, which represents the first step towards the formation of the cytotoxic agent dATP, proceeds at a significantly lower rate as compared to the phosphorolysis of deoxyadenosine. The analysis of the levels of deoxyadenosine and its derivatives in the incubation media reveals that the rates of disappearance of deoxyadenosine and of formation of adenine increase in concert with the reduction of the effect on cell survival.

Topics: Antineoplastic Agents; Colonic Neoplasms; Deoxyadenosines; Humans; Mycoplasma; Pentostatin; Phosphorylation; Tumor Cells, Cultured

We have assessed the intracellular metabolism of 2'-deoxyadenosine in a human colon-carcinoma cell line (LoVo), both in the absence and in the presence of deoxycoformycin, the powerful inhibitor of adenosine deaminase. The combination of 2'-deoxyadenosine and deoxycoformycin has been reported to inhibit the growth of LoVo cells in culture. In this paper we demonstrate that the observed toxic effect is strictly dependent on cell density. In the absence of deoxycoformycin, 2'-deoxyadenosine is primarily deaminated to 2'-deoxyinosine and then converted into hypoxanthine. In the presence of the inhibitor, the deoxynucleoside, in addition to a phosphorylation process, undergoes phosphorolytic cleavage giving rise to adenine. The conversion of 2'-deoxyadenosine to adenine might represent a protective device, emerging when the activity of adenosine deaminase is reduced or inhibited. There is much evidence to indicate that the enzyme catalyzing this process may be distinct from methylthioadenosine phosphorylase and S-adenosyl homocysteine hydrolase, which are the enzymes reported to be responsible for the formation of adenine from 2'-deoxyadenosine in mammals.

Topics: Adenine Nucleotides; Antimetabolites, Antineoplastic; Colonic Neoplasms; Deoxyadenosines; Growth Inhibitors; Humans; Pentostatin; Tumor Cells, Cultured

Different cell lines, 2 from human colon carcinoma (LoVo and HT29) and 1 from Chinese hamster ovary (CHO K-I), were examined to assess the effect of deoxycoformycin (dCF), an inhibitor of adenosine deaminase (ADA), and 2'-deoxyadenosine (dAdo) on their growth. When used alone, neither dCF or dAdo were cytotoxic for the 3 cell lines, while their combination caused inhibition of cell growth, with the following sensitivity: CHO K-I > LoVo > HT29. We studied the pattern of enzymatic activities involved in the metabolism of dAdo in the 3 cell lines. The phosphorylation of dAdo by adenosine kinase appears to play a central role in the toxicity of the deoxynucleoside in combination with dCF. In fact, CHO K-I cells, which are the most sensitive, possess the highest level of this enzyme. Moreover, the cytotoxic effect was almost completely reversed in the 3 cell lines when inhibitors of adenosine kinase, such as 5'-amino-5'-deoxyadenosine and iodotubercidine, were added to the culture medium together with dCF and dAdo. In addition, baby hamster kidney (BHK) adenosine-kinase-deficient (AK-) cells were highly resistant to this treatment. Uptake inhibition of dAdo using dipyridamole also caused reversal of the toxicity. The AMP and deoxyAMP dephosphorylating activities, much lower in the CHO K-I cells, also appear to play a central role in the toxicity of dAdo when adenosine deaminase is inhibited. However, our data suggest that other factors may modulate the toxic effect, such as S-adenosyl-homocysteine-hydrolase inhibition by dAdo at high concentrations.

Topics: Adenosine Kinase; Antimetabolites, Antineoplastic; Cell Division; Colonic Neoplasms; Deoxyadenosines; Dipyridamole; Drug Synergism; Humans; In Vitro Techniques; Pentostatin; Purines; S-Adenosylhomocysteine; Tumor Cells, Cultured

Topics: 5'-Nucleotidase; Adamantane; Adenosine Deaminase; Adenosine Kinase; Adenosine Monophosphate; AMP Deaminase; Animals; Cell Line; Cell Survival; CHO Cells; Colonic Neoplasms; Cricetinae; Deoxyadenosines; Dipyridamole; Humans; Kinetics; Pentostatin; Tumor Cells, Cultured

The mechanism of action of the adenosine analog, neplanocin A (NPC), was investigated in human colon carcinoma cell line HT-29. Cell viability was reduced to 38 and 17% of control by 24-h exposure to 10(-5) and 10(-4) M NPC, respectively. Cytocidal activity was not affected by inhibition of adenosine deaminase with 2'-deoxycoformycin. Concomitant with decreased cell viability was the reduced incorporation of [14C]dThd and [3H]Leu, and to a lesser extent [3H]Urd, into acid-precipitable material. Labeling of rRNA and tRNA during drug treatment for 24 h with [methyl-3H]Met and [14C]Urd revealed that NPC primarily inhibited RNA methylation, and to a lesser extent, RNA synthesis. RNase T2 digests of total RNA indicated that base and 2'-O-methylation were inhibited to approximately the same degree. Metabolites of NPC were measured by reverse-phase high-performance liquid chromatography and it was found that the major drug metabolite was the drug analog of S-adenosylmethionine with little formation of the respective, S-adenosylhomocysteine metabolite. NPC was utilized to a very small degree for RNA synthesis where only 2 and 30 pmol of NPC/A260 were incorporated into rRNA and tRNA after 24-h exposure to 10(-5) and 10(-4) M NPC, respectively. These results indicate that NPC is metabolized to a metabolite of S-adenosylmethionine which is a poor methyl donor for RNA methyltransferases, and that the accompanying decrease in RNA methylation and protein synthesis appears to be related to its cytocidal activity.

Topics: Adenosine; Adenosine Deaminase Inhibitors; Antibiotics, Antineoplastic; Cell Line; Cell Survival; Coformycin; Colonic Neoplasms; DNA Replication; Humans; Kinetics; Methylation; Pentostatin; Protein Biosynthesis; Ribonucleosides; RNA, Ribosomal; Transcription, Genetic