A cytotoxic DNA precursor is taken up selectively by human cancer xenografts.

ImagesFigure 2

The failure of chemotherapeutic agents to be highly effective against most human cancers is widely attributed to drug resistance (Curt et al., 1984;Goldie & Coldman, 1985). Resistance mechanisms are various and may result from selective pressures acting on a genetically unstable population. Resistance does not occur in normal renewal tissues and their sensitivity is dose limiting (Goldie & Coldman, 1985). It has recently been suggested that it might be possible to take advantage of the resistance of cancer cells and the sensitivity of normal cells to anti-cancer drugs (Bagshawe, 1986). Hydroxyurea (HU) which inhibits DNA synthesis probably through its action on ribonucleotide reductase (Ackerblom et al., 1981) is relatively ineffective against most solid cancers and resistance readily develops (Ariel, 1970). It was therefore suggested that treatment with inhibitors of DNA synthesis should cause more marked inhibition of DNA synthesis in normal renewal tissues than in resistant cancers. If so, then it might be possible to incorporate precursors of DNA that are cytotoxic, or suitable for scintigraphic imaging, selectively into tumour cell DNA. It was further suggested (Bagshawe, 1986) that this approach might be explored using the pyrimidine analogues 5-iodo-2'-deoxyuridine (IUdR) and 5-bromo-2'-deoxyuridine (BUdR) which differ from thymidine, the normal pyrimidine base, only by substitution of a halogen for the 5-methyl group. They compete with thymidine for phosphorylation and incorporation into DNA (Prusoff, 1959;Djordjevic & Szybalski, 1960). IUdR is rapidly dehalogenated unless incorporated into DNA but IUdR in DNA is retained until the cell divides or dies. IUdR and BUdR are known radioand photo-sensitisers (Djordjevic & Szybalski, 1960) and 12I-IUdR is a potent cytotoxic agent (Hofer, 1980). Preliminary experiments to test the hypothesis were performed in nu/nu mice carrying a human choriocarcinoma xenograft (CC3) (Figure 1 a-h). Group 1 (Figure la) received only 125I-IUdR and tissues excised 24h later showed, as have previous studies (Shuhmacher et al., 1974;Hampton & Eidinoff, 1961) that uptake of 125I-IUdR was -4 times greater in small intestine and colon than in tumour. When HU was given before 125I-IUdR (group 2, Figure lb) the total counts for intestinal tissues were substantially reduced but tumour counts were not reduced, indicating differential sensitivity to HU and suggesting that DNA synthesis continued in the tumour when it was suppressed in normal renewal tissues.
Methotrexate (MTX), which reduces thymidine synthesis through its anti-folate action, was also given to CC3 bearing mice ( Figure Id). MTX produced a less marked increase in uptake of 125I-IUdR than 5FU in the dosages employed, but tumour uptake was again increased relative to that by normal tissues. We had therefore shown that a fluoropyrimidine and a folate antagonist increased uptake of 1251_ IUdR in both tumour and normal renewal tissues.
In the next study (group 5, Figure le) 5FU and HU were given together before and during exposure of the mice to 125I-IUdR. There was a reduction in uptake by all tissues compared with group 3 (Figure ic) which received only 5FU and 125I-IUdR but the reduction in tumour uptake was less marked so that the mean tumour to colon ratio was 0.91. A similar effect occurred when MTX and HU were given (group 6, Figure If) but the effect of reducing normal tissue uptake of 125I-IUdR was greater and the mean tumour:colon ratio was 4.5.
Since uptake of IUdR is restricted to cells in S phase or engaged in unscheduled DNA synthesis (Lewensohn et al., 1982), a high proportion of tumour cells is likely to be labelled only by repeated administration. This was studied first by giving HU and '25I-IUdR on each of 3 successive days (Figure ig). Intestinal tissue counts were not higher than after the same drugs given once (Figure lb) but counts in the tumour were increased giving a mean tumour:colon ratio of 2.4. When 125I-IUdR was given after HU and 5FU on 3 successive days a mean tumour:colon ratio of 8.1 was obtained. The proportion of total administered dose retained in the tumour was 0.7%g-1 24h after the last injection of  These studies achieved a selective uptake of 125I-lUdR in a human cancer xenograft in mice. Prolonged retention of 125I-IUdR by the tumours was indicated by the data from groups 7 and 8 ( Figure Ig and 1h), and this was consistent with incorporation of 125I-IUdR into DNA. Excretion of 1251 was not complete by 24h so that free 1251 or 1251 bound non-specifically to protein (Prusoff, 1963) contributed to both tissue and tumour radioactivity when measured by gamma counting the digested tissues.
The intracellular location of 125I-IUdR is particularly relevant to its potential cytotoxicity; within the nucleus it is highly cytotoxic but toxicity is low when 125I-1UdR is confined to the cell membrane (Hofer, 1980). Autoradiographs were therefore obtained. Figure 2  Counts g-1 min-' Figure 1 Tissue and tumour uptake of 125INUdR. The histograms show tissues in the same order for each study as indicated in a. Bars indicate 1 s.d. Groups of 4 or 5 nu/nu mice carried xenografts of a human choriocarcinoma (CC3) 0.5-1.5 cm diameter on one flank. The tumour was obtained at surgery from a patient who had no prior chemotherapy; it has been passaged for 6 years, secretes human chorionic gonadotrophin and maintains its original morphological and growth rate characteristics. The mice weighed 20-25g and received standard feed and water ad libitum with added potassium iodide. 1.25pCi 125I-IUdR (5mCigg-1, Amersham International, UK) was given to each mouse (except group 8 detailed below). Other drugs (see below) were given by i.p. route 24h before 125I-IUdR and the same dose was repeated at the time of receiving 125I-IUdR. The mice were killed by cervical dislocation 24h after 125I-IUdR administration, the organs dissected out immediately,. blotted, weighed, digested in 7 M KOH for 12 h and the gamma emission counted. Mice received the following agents: group 1 (a), saline only; group 2 (b), HU 50mg kg-1; group 3 (c), SFU 20mg kg-1; group 4 (d), MTX 5 mg kg-1; group 5 (e), HU 50 mg kg-1 + SFU 20 mg kg-1; group 6 (f), HU 50 mg kg-I + MTX S mg kg-1; group 7 (g), as group 2 on 3 successive days; group 8 (h), HU 50mgkg -1+SFU 20mgkg -1+MTX 5mg kgi.p. 30min before O0pCi 1251-IUdR i.v.; HU was repeated 1 h and folinic acid given 5 mg kg-1 2 h after IUdR on 3 successive days.  Figure 1 and group 9 under Figure 2. The tumour:colon tissue count ratio is derived from the data shown in Figure 1. Additional mice were included for autoradiography in groups 1, 2, 3, 6, 7; protocols were identical except that they received a large (lSyCi) dose of 1251-IUdR.
To perform nuclear grain counts the slides of tumour and colon tissue were examined under x 100 oil immersion objective. An eyepiece graticule comprising 25 points contained within a circle and divided into 4 quadrants (Graticules Ltd., Tonbridge, Kent) was used to select tumour nuclei for counting. Where a point overlay or touched a nucleus the grains within the nucleus were counted. Where a point did not fall directly over or touch the edge of a nucleus, the nucleus of the first cell on an imaginary straight line to the left was used. Where this line crossed the perimeter of the graticule no point counting could be carried out. Different fields were examined until a total of >400 nuclei had been counted. The number of grains confined within an area bounded by the nuclear membrane were counted. Grains outside the nuclear membrane were not included. Nuclear grain counts were performed on sections of colon by examining at least 10 crypts cut longitudinally so that the distribution of grains within crypts could be assessed. Starting at the mid-point of the base of the crypt and ascending on either side to the tip, nuclear grains were counted. Mean grain densities per nucleus were calculated from at least 400 nuclei.
--- Figure 2 Autoradiographs of CC3 tumour (a) and colon (b) from a nu/nu mouse which received 5FU 20mg kg-1 + HU 50mg kgi.p. followed after 30min by 1251 IUdR 50pCi i.v. on 5 alternate days (group 9, Table I). The mice were killed on day 11. Colon from a mouse which received 5FU and 251IIUdR but no HU (group 3) is shown in (c) for comparison.
Photomicrographs were taken at an original magnification x 240, enlarged x 3.
Pieces of liver, colon and CC3 tumour xenograft were fixed in 10% buffered formalin for 24h. Femoral bone pieces were decalcified in an EDTA formalin mixture (80g EDTA in 950ml of distilled water plus 50ml formalin) for 16h and then post-fixed in 10% buffered formalin for 8h. Tissues were processed through a graded alcohol sequence and an inhibisol/chloroform mixture to paraffin wax. Sections were cut at 4pm and mounted on subbed slides (dipped in 0.5%/ gelatine in 0.05% potassium chromium sulphate in distilled water). Slides were covered by Kodak ARIO stripping film and autoradiographs were prepared by standard methods.
graphs of tumour (group 9) and colon (group 9 and group 3 for comparison). The mean nuclear grain count ratio for tumour: colon was 26.2 in group 9. Compared with total tissue counts nuclear grain counts consistently suggested a more favourable distribution of 125I-IUdR (Table I). In the 5 groups studied by both techniques the mean nuclear grain count ratios were 5.4-14.0-fold higher than the respective tissue count ratios. Cumulative frequencies of nuclear grain counts were determined for colon and tumour and these also confirmed that the number of labelled colonic nuclei and the number of grains within them were reduced by the addition of HU to 125I-IUdR but the corresponding numbers in tumour nuclei were little changed.
Limited studies using cytosine arabinoside (CA) in place of HU and studies on a human colorectal carcinoma xenograft have given comparable results to those described here with the CC3 tumour but with slow growing tumours 125I-IUdR uptake is likely to be lower.
No attempt was made in these early studies to ensure the CC3 tumour was resistant to HU nor to optimise the dosage and timing of the drugs used. Nevertheless, the tumour: normal tissue distribution of 125I-IUdR has been modified in this xenograft model to one that is potentially favourable for diagnostic and therapeutic purposes by blocking normal tissue uptake with HU and, in addition, giving agents which increase 125I-IUdR uptake by uninhibited cells. Further enhancement of tumour uptake of 1251-IUdR may be possible by inhibiting thymidine kinase with 5'-aminothymidine (Fischer et al., 1986). The role of HU or CA in this approach is a reversal of their normal role as anti-cancer agents.
Refinement of the present technique might provide a basis for estimating cell deaths as they occur spontaneously, or as a result of therapy in animal tumour models and perhaps in man. Immunoscintigraphic methods are used clinically to identify sites of drug resistant tumour (Bagshawe, 1985); 131 -IUdR or 1231-IUdR may prove superior to antibody directed isotopes for some tumours because retention of radioactivity in blood, liver and lungs is relatively low.
Since IUdR and BUdR are known to act as radiosensitisers and photosensitisers their selective uptake by tumour cells may prove advantageous. Their possible role as sensitisers to alkylating agents (Prusoff, 1963) or other chemotherapeutic drugs requires re-investigation. 125I, 1231 and ""Br are characterised by Auger electron emission which greatly enhances their therapeutic potential (Hofer 1980, Lemotte & Little, 1984. The penalty for achieving a selective uptake of a thymidine analogue into tumours is a period of inhibition of DNA synthesis and consequent cell loss from normal tissues. The ultimate equation is the therapeutic:toxicity ratio which reflects total cell losses in tumour and normal tissues resulting from the drugs used to achieve the selective distribution of the analogue as well as the analogue itself.