Insea Biochem., ,972, 2, 87-93. [Scientechnica (Publishers) Ltd.] 87

THE EFFECT OF VINBLASTINE SULPHATE ON THE

INCORPORATION OF [2-~'C]GLYCINE INTO HOUSEFLY DNA

STEVE MILLER, JEANETTE M. COLLINS, AND LAWRENCE DEAN FRENKEL*

Technical Development Laboratories, Laboratory Division, Center for Disease Control, Health Services and Mental Health Administration, Public Health Service, U.S. Department of Health,

Education, and Welfare, Box 2167, Savannah, Ga. 3,4o2, U.S.A.

(Received i 3 May, I97I)

ABSTRACT

The synthesis of DNA was studied in houseflies 48 hours after emergence in the presence and absence of vinblastine sulphate (VLB).

In all cases administration of the alkaloid inhibited the incorporation of [2-x4C]glycine into DNA. The site of inhibition has not been established.

Analysis of the bases in DNA revealed that the specific activities of DNA purines were decreased (relative to control values) more than those of DNA thymine.

VLB caused sterility in the female flies but not in the male flies. A method is described for preparing a reproducible preparation of high

quality DNA from houseflies.

NUMEROUS chemotherapeutic agents are used for the treatment of neoplastic disease. These include alkylating agents, antimetabolites, and antibiotics. The isolation from the periwinkle plant (Vinca rosen Linn.) of alkaloids with antitumour activity added a new type of compound to this group of chemicals used in cancer chemotherapy. The vinca alkaloids are used for the treatment of Hodgkin's disease and choriocarcinoma (Johnson, Armstrong, Gorman, and Burnett, 1963). Evidence of interference with DNA synthesis in cells exposed to vinblastine sulphate (VLB) has been reported. Tanko (1962) demon- strated that the treatment of ascites lymphoma NK/Ly in mice with VLB produced a decrease in the amount of DNA between 6 and 96 hours after administration of the alkaloid. VLB inhibits the incorporation of labelled precursors into nucleic acids of rat bone-marrow in vitro (Palmer, Livengood, Warren, Simpson, and Johnson, 196o ).

Since some antimetabolites, alkylating agents, and antibiotics produce sterility in one or both sexes of adult houseflies (LaBrecque, Adcock, and Smith, 196o; Kohls, Lemin, and O'Connell, 1966; Fye, 1967) ' studies were undertaken to determine whether the administration of VLB would produce sterility in the housefly and have an effect on the de novo synthesis of DNA. The conversion of [2-14C]glycine into the base com- ponents of DNA was used as a measure of the de novo route, since it has been previously shown that this simple precursor is utilized in the biosynthetic pathway leading to the nucleic acids (Miller, 1969; Miller and Collins, 197o ) .

Several procedures have been described for the isolation of DNA from Drosophila flies (Mead, 1964; Hastings and Kirby, 1966 ). However, no detailed account is available for the isolation of DNA from houseflies. The method described here has been modified

* Resident Pediatrician, New York Hospital, New York, N.Y., U.S.A.

88 MILLER AND OTHERS Insect Biochem.

pr imar i ly f rom the procedures which have been repor ted for D N A isolation by M e a d

( i964) and M a r m u r ( i963).

MATERIALS AND M E T H O D S

CHEMOSTERILANT STUDIES Adult houseflies, Musca domestica L. (NAIDM) strain, were reared as larvae on a CSMA media

and maintained in our insectary at 27 ±2 ° C. After pupation the pupae were removed from the sand and placed in cages. Flies emerging in the first 6-hour period were discarded, and flies emerging in the next 6-hour period were collected and anaesthetized.

When treating both sexes, xoo male and ioo female flies were placed together in a cage and fed a treated diet consisting of powdered milk/sucrose (i : I) with o ' i per cent VLB for 48 hours. The second and third cultures were offered the treated diet for 72 and 96 hours respectively. Observa- tions were made on the relative toxicity of the alkaloid. At the end of these periods the flies were placed on a diet consisting of powdered milk/sucrose (I : i). Oviposition medium was offered at the beginning of the sixth day, and at 24-hour intervals the medium was checked for egg deposition. If eggs were present they were collected and placed on larval medium and observed for fertility. Control flies receiving no alkaloid were used as standards. Each test was repeated twice and results were compared with those of an untreated control.

To determine whether VLB sterilized one or both sexes another series of experiments was run, in which flies of each sex that had fed for 48 hours on a treated diet were crossed with flies of the opposite sex that had fed on treated and untreated diets. The combined flies were fed untreated food for the remainder of the testing period.

INSECT MATERIAL Five hundred female flies 48 hours after emergence, which had been fed on a treated diet,

were injected laterally through the sternum in the metathoracic region with o ' i ~tc. of [2-14C] - glycine (242,ooo c.p.m.) in i Id. of distilled water. The flies were placed on a sugar diet. Six hours after injection they were anaesthetized and frozen. Control flies receiving no alkaloid were injected in the same manner and frozen.

CHEMICALS Vinblastine sulphate was a gift from Eli LiUy Co.* Pancreatic RNase was obtained from the

Sigma Chemical Co. and T t RNase from the Calbiochem Co. [2-t4C]Glycine was purchased from New England Nuclear Corporation.

DNA ISOLATION The frozen female houseflies (500) were homogenized in 7S ml. of cold saline/citrate/EDTA

(o'I5 M NaCl+o 'o I5 M sodium citrate+o'o5 M ethylenediaminetetra-acetate, pH 8) in an all-glass homogenizer. All operations were carried out at 3-5 ° C. unless otherwise indicated. The homogenate was centrifuged at zooo g at 5 ° C. for io minutes and the supernatant discarded. This step was repeated four times to remove contaminating cytoplasmic RNA. After the final centrifugation the nuclear pellet was suspended in 40 ml. of o'xo M NaCI and stirred for xo minutes. To the suspension was added an equal volume of 5 per cent Aerosol OT, and the nuclei were lysed for 2"5 hours at 5 ° C. with agitation. Lysis was completed by adding sodium deoxycholate, final concentration o'o4 per cent, and stirring for io minutes. All subsequent steps could be carried out more efficiently after the minute quantity of deoxycholate was added. The mixture was made i M with respect to NaCI, stirred for an additional io minutes, and centrifuged at io,ooo g for 2o minutes. The supernatant was shaken with an equal volume of ehloroform/isoamyl alcohol (24 : x v/v) in a ground-glass stoppered flask for I5 minutes. An emulsion was formed which separated into three layers after a 15-minute centrifugation at

* The use of trade names is for identification purposes only and does not constitute endorsement by the Public Health Service or the U.S. Department of Health, Education, and Welfare.

I972 , 2 METABOLIC EFFECTS OF VINBLASTINE 8 9

Io,ooo g. The upper aqueous phase containing D N A was carefully pipetted into a beaker. Two volumes of cold ethanol were added to the D N A solution. The precipitated white D N A fibres were gathered by winding on a glass rod and the excess fluid was removed by gently pressing on the side of the beaker. The precipitate was transferred to zo ml. of standard saline/citrate (o" I5 M NaCl-ko'ox5 M sodium citrate, p H 7"o) and gently removed from the rod by swirling it back and forth. The chloroform/isoamyl alcohol deproteinization was repeated by shaking for 15 minutes. The D N A was precipitated with z volumes of ethanol as before and the precipitate was wound on a glass rod and dissolved in xz ml. of standard saline citrate. A mixture of heated (xo minutes at lOO ° C.) pancreatic RNase and T I RNase in o-x5 M NaCI was added to a final concentration of 1"o mg. per mL and zo units per nd. respectively. The solution was stirred and incubated at 37 ° C. for 4o minutes. The suspension was shaken for 15 minutes with an equal volume of chloroform/isoamyl alcohol in a ground-glass stoppered flask. The resulting emulsion was broken by a xs-minute centrifugation at xo,ooo g. The aqueous phase containing the D N A was removed by a pipette. The D N A was precipitated from the aqueous phase by slowly adding z volumes of cold absolute alcohol The precipitate was wound on a glass rod and dissolved in lO ml. of standard saline/citrate. Pronase (treated previously to denature DNase) was added to a final concentration of x mg. per ml. The mixture was incubated at 5 °o C. for z hours. After the incubation MgCla was added to a final concentration of o'7 M and stirred for 3o minutes at room temperature. After centrifugation at xz, i o o g for xo minutes the resulting precipitate was dis- carded. The D N A was precipitated with z volumes of ethanol. The precipitate was wound on a glass rod and dissolved in 8 ml. of standard saline/citrate. The chloroform/isoamyl alcohol deproteinization was repeated by shaking for x5 minutes. The D N A was precipitated with z volumes of ethanol, and the precipitate was collected. The D N A was dissolved in 9 ml. of dilute saline/citrate (o 'oi5 M NaClq-o'oox5 M sodium citrate, p H 7"o). One ml. of acetate/EDTA (3"o M sodium acetateq-o.ooi M EDTA, p H 7"o) was added and stirred for 6o seconds on the Vortex mixer. The D N A was precipitated with o'54 volume of isopropanol. The precipitate was wound on a glass rod and dissolved in 5 ml. of standard saline/citrate. The D N A was precipitated by adding z volumes of cold ethanol, and the resulting fibres were collected on a glass rod. The D N A was dissolved in 5 ml. of o'o7 M NaCI and centrifuged at zooo g for io minutes. The slight residue was discarded. The amounts of D N A and RNA were deter- mined.

CHEMICAL DETERMINATIONS

The ribose in RNA was determined by the orcinol method (Schneider, i957) and D N A was determined by the diphenylamine reaction (Disehe, I955). Protein was determined hy the method of Lowry, Rosebrough, Farr, and Randall (195 x); bovine serum albumin was used as the standard.

ISOLATION OF PURINE AND PYRIMIDINE BASES FROM D N A

D N A samples ( i .8-4. 3 nag.) were hydrolysed with 70 per cent perctdoric acid ( ioo ° C. for x hour). Excess perchloric acid was removed as KCIO4, and the bases in the supernatant fraction were separated by cellulose thin-layer chromatography with Wyat t solvent, which consisted of z M HCI and 65 per cent isopropanol. After development the compounds were located by ultra-violet light and identified by comparison with known bases. The samples were eluted with o'1 M HCI. The spectra of the bases were determined against appropriate thin-layer blanks in a

Cary I4 recording spectrophotometer. Concentrations of bases were calculated by using estab- lished extinction coefficients. In previous work (Miller, 1969) adenine, guanine, and thymine showed no change in specific activity upon rechromatography, therefore only cytosine was sub- jected to further purification. Cytosine was resolved from radioactive components by high- voltage electrophoresis on cellulose thin-layer plates. Sodium formate buffer (pH 3"4, ionic strength o.x) was used for development with a potential difference of 6o0 V. The specific activity of cytosine was redetermined.

RADIOACTIWTY MEASUREMENTS

Aliquots of the eluates from plates were added to vials containing scintillation solution (Hall and Cocking, 1965). The radioactivity of each base was measured in a Nuclear Chicago Mark I Scintillation Spectrometer and an external standard for quenching corrections was used.

9 ° MILLER AND OTHERS Insect Biochem.

RESULTS In isolating DNA from houseflies the major difficulty encountered was contamination

of DNA by RNA and protein. Several methods were used for the isolation and the method presented gave the highest yield of DNA. The sequence of steps in the purifi- cation of DNA was chosen to give the highest degree of deproteinization and inhibition of nucleases. The action of deoxyribonuclease was minimized by adding EDTA to the buffer at pH 8.o. DNA was released from the nuclei by 5 per cent Aerosol OT. Aerosol OT promoted dissociation of the DNA-protein complex. The removal of RNA from

Table I.--INcoRPORATION OF [2-t~C]GLYcINE INT0 BASF_S OF DNA

TISSUE *

{ousefly ovary {ousefly

SPECIFIC ACTIVITY OF BASE (c.p.m. per pmole)

t Adenine

I 38,1Ol 16,752

Guanine

3o,381 14,294

Cytosine

478 562

Thymine

15,511 3681

* Flies sacrificed at 48 hours.

Table 1/.--EFFECT OF VINBLASTINE SULPHATE ON HOUSEFLY DNA

ADDITION *

None VLB

DNA CONTENT t (mg. per g. housefly)

0"38 0"25

EFFECT OF VLB ON INCORPORATION OF [2-1'C]GLYCINE INTO DNA

(c.p.m. per vmole)

Adenine Guanine

16,752 14,294 6235 5504

Thymine

3681 1667

* Vinblastine sulphate (O'I per cent) added to food at time of emergence. Flies sacrificed after 48 hours.

I" The values represent the average obtained in 3 separate experiments.

protein was accomplished by deproteinization prior to ribonuclease treatment. Pan- creatic and T 1 ribonuclease were used to digest RNA. After four cycles of deproteiniza- tion by means of chloroform/isoamyl alcohol, DNA was essentially free of RNA and contained no detectable protein. The chloroform/isoamyl alcohol mixture not only removed the protein but also lipid material which interfered with the purification of DNA. Since uracil could not be detected on the thin-layer chromatograms of the acid hydrolysate of DNA, this indicated further that no RNA was present. A typical DNA absorption (E=so/E=so ratio of 2.o) was obtained which indicated that the DNA was of high purity. The molar percentages (based on 3 determinations) of adenine, thymine, guanine, and cytosine were 31"28, 3z'28, I8"25, and x9.2I respectively.

It has been shown that the housefly ovary synthesizes the constituents of DNA and RNA from relatively simple precursors (Miller and Perry, z968; Miller and Collins, z97o ). The data presented in Table l show clearly that [2-*4C]glycine is also incorporated to a considerable extent into adenine, guanine, and thymine but to a negligible degree

I972, 2 METABOLIC EFFECTS OF VINBLASTINE 91

into cytosine of 48-hour-old houseflies. The incorporation into purine ovarian DNA is 2.I-2. 3 times greater and the incorporation into thymine is 4"2 times greater than it is into housefly purine DNA. The marked difference in incorporation between the ovary per se and the housefly indicates rapid DNA synthesis in the developing ovary.

The effect of VLB on DNA synthesis in the housefly was examined. The age of the houseflies was chosen on the basis of the rate of DNA synthesis, as shown by the specific activity of the DNA bases and inhibition of oviposition. Of the various concentrations of VLB added to food, o.i per cent was not unduly toxic. Therefore, the houseflies after emergence were kept for 48 hours on a diet with and without o" i per cent of the alkaloid. A longer oral administration of the alkaloid at o. i per cent resulted in a decrease in oviposition and an increase in mortality. The effect of VLB on the incorporation of [z-t4C]glycine into the bases of DNA is shown in Table II. There is a differential effect on labelling of the purines and thymine of DNA. The specific activity of the DNA thymine of houseflies exposed for 48 hours to VLB was decreased to about 45 per cent of the value of the control. However, at that time the specific activity of the DNA purines decreased to about 37 per cent of the value of the control. There was essentially equal inhibition of the incorporation of radioactive glycine into both guanine and adenine. The values presented represent the average of at least 3 separate incorporation experiments. Reproducibility in different experiments was remarkably close. There was also a significant decrease in DNA content in houseflies administered VLB. These results suggest that VLB may be particularly affecting the formation of DNA purines. In the biosynthetic pathways leading from simple precursors, such as glycine, to the polymeric DNA there are many intermediate reactions which might be inhibited by the alkaloid.

When certain antimetabolites and alkylating agents are given to adult houseflies in food at a concentration of o.o5-i per cent, these compounds produce sterility in one or both sexes. Some sterilants interrupt or prevent normal oviposition, whereas others cause the deposition of non-viable eggs. VLB at a concentration of o.I per cent caused sterility in the females but not in the males. Females given treated food for 48 hours showed a 94-97 per cent inhibition of egg-laying and no toxic effect, whether exposed to males fed treated or untreated food. The deposited eggs were viable. The length of time that the houseflies were fed with o.i per cent of the alkaloid was increased to 72 and 96 hours. This increase resulted in a complete inhibition of egg production accom- panied by an increase in mortality. Two weeks after emergence the mortality of females given treated food for 72 hours was 65 per cent. Females given untreated food oviposited freely when exposed to males fed treated or untreated food, thus demonstrating that sterility caused by VLB occurs in the female fly only.

DISCUSSION The studies undertaken in this laboratory are directed towards an understanding of

nucleic acid synthesis in the housefly. From preliminary investigations it has been established that nucleotides originate either de novo or from preformed purines and pyrimidines. The relative contributions of these pathways are important in selecting inhibitors that may be used in insect control. The demonstration, as reported in this paper, that VLB is a chemical sterilant for the female housefly led to a study of the biochemical activity of the alkaloid. Although differences in biochemical mechanisms

9 2 MILLER AND OTHERS Insect Biochem.

of action may exist among the different vinca alkaloids, as well as between cellular systems, the data reported here compare with the observations of others concerning the action of VLB. Richards and Beer (1964) reported the effect of VLB on the [14C]formate incorporation into nucleic acids of rat thymus cells in vitro. They showed in this cell system that VLB was affecting particularly the formation of DNA purines. The specific activities of DNA adenine and DNA guanine of cells incubated with VLB were decreased below control levels to a greater extent than were specific activities of DNA thymine. Luyckx and Van Lancker (1966) showed that VLB interferes with the incorporation of [l'C]orotic acid and [3I-I]thymidine into deoxyribonucleic acid 24 hours after partial hepatectomy, a time at which DNA synthesis occurs at a maximal rate in , ivo. Creasey and Markiw (1964) presented evidence that VLB is able to inhibit two distinct bio- chemical processes: the synthesis of DNA and the turn-over of sRNA. The uptake of thymidine into DNA of Ehrlich ascite cells in vitro was affected by moderate concentra- tions of VLB. [2-1*C]Glycine incorporation into DNA can be considered to be a reliable index of DNA synthesis. The radioactive precursor enters intracellular pools; however, fluctuations in these pools can stimulate or reduce incorporation without corresponding changes in the actual rate of DNA synthesis. The houseflies were sacrificed 48 hours after emergence, which allowed sufficient time for feeding and for the maximal rate of DNA synthesis to occur, as shown by incorporation studies. The data presented indicate equal inhibition by VLB of the incorporation of radioactive glycine into both guanine and adenine of DNA. There is also a differential effect on labelling of the purines and thymine of DNA by VLB. This pronounced inhibition of the incorporation of the precursor into DNA indicates the presence of a site of inhibition on the pathway of purine nucleotide formation de novo, but it is not certain whether this is a direct effect. Glycine is a precursor in the de novo synthesis of inosinic acid and subsequently of other purine nucleotides. It is also possible that VLB may not interfere with the biosynthesis of inosine but may inhibit a reaction in the pathway leading from ribo- nucleotide to DNA. In order to study further the site of inhibition it is necessary to study the effect of the alkaloid on the utilization of 4-amino-[2-t4C]-imidazole-5-carboxamide in which group is the 1'C. The effect of VLB on nucleic acid synthesis may not be at the primary site of alkaloid activity. The alkaloid may cause a biochemical disturbance else- where; it may interfere with a protein necessary for DNA synthesis but not interfere with thymidylic kinase and DNA polymerase. It also remains to be established whether VLB interferes with DNA replication and if this DNA is necessary for the synthesis of RNA. If the newly synthesized DNA is not of immediate importance in the synthesis of RNA no change in the RNA pattern will be observed. Preliminary experiments indicate that the action of VLB in the housefly appears to be similar but more specific than the action of actinomycin D. In intact cells, as well as with purified enzymes, actinomycin D inhibits selectively the DNA-directed synthesis of RNA but not of DNA, the template is the same for both syntheses. The vinca alkaloids and the actinomycins are among the most potent anti-tumour substances known. More detailed studies are now in progress with actinomycin D and VLB in houseflies.

The difference in the incorporation of [2-1*C]glycine into housefly ovary per se and into the housefly indicates the difference in organ-specific rates of glycine utilization and conversion. Investigations of VLB in other biochemical systems demonstrated that the alkaloid interferes with the proliferation of rapidly growing tissues. The ovaries from flies administered VLB were smaller than those of flies given normal food. The

t972, 2 METABOLIC EFFECTS OF VINBLASTINE 93

inhibition by VLB of D N A formation results in a critical failure of the ovaries to develop.

As these agents are used more frequently in human disease and as viability is main- tained to and beyond the reproductive years, the question of human sterility may become increasingly important.

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Key Word Index: Vinblastine sulphate, [2-x~C]glycine, Musca domestica, insect biochemistry, de novo synthesis.