Showing papers in "Cancer Research in 1972"

Synthesis of -fetoprotein by liver, yolk sac, and gastrointestinal tract of the human conceptus.

Abstract: The synthesis of serum α-fetoprotein was studied in 16 human embryos and fetuses between 4.2 and 18 weeks of gestation by incubation of selected tissues in 14C-labeled amino acids followed by immunoelectrophoresis of the culture fluids and radioautography. Relatively large amounts of radioactive α-fetoprotein were found in each of the liver cultures and in cultures of the developing yolk sac. Smaller amounts of labeled α-fetoprotein were observed in almost all of the gastrointestinal tract cultures. Labeled α-fetoprotein was formed in the kidney cultures from 1 of 9 conceptuses and in only 1 of 14 placentas cultured. None of the cultures of lung, thymus, pancreas, skeletal muscle, amnion, chorion, or blood produced detectable amounts of α-fetoprotein.

Liver microsomal metabolism of aflatoxin B 1 to a reactive derivative toxic to Salmonella typhimurium TA 1530.

Abstract: Summary A reduction in the survival of Salmonella tryphimurium TA 1530 was observed when the bacteria were incubated with aflatoxin B 1 , rat liver microsomes, and a reduced nicotinamide adenine dinucleotide phosphate-generating system. The lethality appeared to depend on the formation of a metabolite of aflatoxin B 1 by a mixed-function oxygenase system. The killing was very rapid; only 1% of the bacteria were able to form colonies after 2 min of incubation with large amounts of microsomes and aflatoxin B 1 . Attempts to separate the toxic metabolite from the microsomal system have not been successful. Toxic metabolites for S. typhimurium TA 1530 were also formed if aflatoxin B 1 was replaced by either aflatoxin G 1 or sterigmatocystin in the microsome-mediated toxicity assay. Except for aflatoxicol, the derivatives that were tested either had much less activity or were inactive. The livers from a number of other species of rodents and a single autopsy sample of human liver also were active in the microsome-mediated aflatoxin B 1 toxicity assay. The addition of RNA or DNA to the incubation mixture inhibited the killing of the bacteria. The RNA (which was reisolated after its incubation with aflatoxin B 1 ), liver microsomes, and a reduced nicotinamide adenine dinucleotide phosphate-generating system showed a low, broad absorption, with a maximum at 366 to 370 nm. This high wavelength absorption was not removed by Sephadex G-10 chromatography of the RNA or by extraction procedures and appeared to be attributable to covalently bound aflatoxin B 1 toxicity assay. The formation of the conjugated RNA was dependent on reduced nicotinamide adenine dinucleotide phosphate and was inhibited by the addition of aniline; the amount formed was a function of the activity of the mixed-function oxygenases in the incubation mixture. On the basis of the data presented, it is tentatively suggested that the derivative that is toxic to S. typhimurium TA 1530 and the one that reacts with nucleic acids are identical. The possible relationship of this derivative to the hepatocarcinogenicity of aflatoxin B 1 is discussed.

Epidermodysplasia verruciformis as a model in studies on the role of papovaviruses in oncogenesis.

Abstract: Summary Epidermodysplasia verruciformis is a skin disease caused by a generalized infection by verruca virus in which the verrucous lesions usually change into tumors, most frequently Bowen9s carcinoma. Lesions from a case in which papovavirus was evident were transmitted to a healthy person, the virus being found in cell nuclei in the wart lesions and also in lesions with some signs of atypia. This fulfilled the condition for recognition of the virus, demonstrated by electron microscopy, as being causatively involved in the verrucous lesions in epidermodysplasia verruciformis and also in the initiation of the morbid process. The virus could not be demonstrated in lesions showing distinct signs of cancer.

The role of glutamine in the oxidative metabolism of malignant cells.

Abstract: Summary Experimental evidence is presented indicating a very good correlation between the activity of mitochondrial glutaminase and the rate of respiration of tumor mitochondria in the presence of glutamine. K m measurements for glutamine of glutaminase in intact, coupled mitochondria isolated from Morris hepatomas showed a correlation between the increase of the affinity of the enzyme for the substrate and the rate of growth of the tumors. Comparison of the rate of oxygen consumption by isolated mitochondria in the presence of glutamine and two other physiological substrates, glutamate and pyruvate, and the measurement of CO 2 production by intact tumor cells in the presence of glutamine and glucose indicated that glutamine is one of the most important substrates in the oxidative and energy metabolism of rapidly growing malignant cells in vivo . If the mitochondria are able to respire in the presence of low concentrations of glutamine (below 1 mm), they do not swell in the isoosmotic solution of ammonium glutamate, which indicates the relative impermeability of the mitochondrial membrane for glutamate. Measurement of the distribution of glutamate during glutaminase activity revealed a high accumulation of this metabolite in the mitochondrial compartment. Because glutamate is a good reductant for NAD(P) + , it was assumed that this is one of the factors in the maintenance of a great difference in the oxidation-reduction state between intra- and extramitochondrial pyridine nucleotides; this difference is important for mitochondrial respiration and energy production.

Action of Camptothecin on Mammalian Cells in Culture

Abstract: Summary Camptothecin (CN) is active against several experimental tumors and has also been studied clinically. We report here the effect of CN on L1210 cells and asynchronous and synchronous DON cells in culture. CN was toxic both to L1210 cells (0.06 µg/ml, 2-hr exposure) and DON cells (0.15 µg/ml, 1-hr exposure), and it inhibited DNA and RNA synthesis more than it inhibited protein synthesis. CN was more cytotoxic to DON cells in S phase than cells in G1 or G2, although it inhibited DNA and RNA synthesis of L1210 cells and asynchronous DON cells almost equally. When asynchronous DON cells were exposed to CN for 30 min and CN was then removed, RNA synthesis was no longer significantly inhibited, but the inhibition of DNA synthesis persisted. The survival patterns of synchronous DON cells were closely related to DNA synthesis inhibition but not to RNA synthesis inhibition. These results collectively suggested that the marked effect of CN on DNA synthesis appeared to be one of the primary determinants of its cytotoxicity. Since no significant effect was observed on the enzymes involved in DNA synthesis, DNA synthesis inhibition by CN may be in part due to its effect on the DNA template. The interaction between CN and DNA was detected by melting point determinations. CN did not block the progression of mitotic cells into S phase. At 100 µg/ml, CN prevented the progression of S phase cells into G2; at 1 µg/ml, some cells did leave S and proceeded into G2. Cells in G2 were blocked from moving into mitosis even at 0.01-µg/ml doses of CN. Thus, the progression of late S or early G2 cells into mitosis was most sensitive to the drug.

Enzymatic Metabolism of Cyclophosphamide and Nicotine and Production of a Toxic Cyclophosphamide Metabolite

Abstract: Summary Cyclophosphamide is converted by enzymes of mouse liver into two metabolites. Production of the first (aldophosphamide), which is uncharged, requires TPNH, is inhibited by CO, and is accomplished predominantly by the microsomal fraction. With the microsomal enzyme, the Km for cyclophosphamide is 0.5 mm; nicotine, atropine, ephedrine, apomorphine, and cocaine are potent inhibitors. Phenobarbital, cytochrome c, 2-diethylaminoethyl-2,2-diphenylvalerate, and some steroid hormones also inhibit the reaction. Aldophosphamide is very toxic, as judged by inhibition of clone formation of human epidermoid carcinoma No. 2 cells and by toxicity to L1210 leukemia cells. The initial metabolite is further converted to 2-carboxyethyl N,N-bis-(2-chloroethyl)phosphorodiamidate (carboxyphosphamide) by an enzyme in the soluble portion of the cell. This enzyme can be replaced by purified aldehyde oxidase (aldehyde:oxygen oxidoreductase, EC 1.2.3.1). Carboxyphosphamide, which has little or no antitumor effect, is much less toxic to clone formation of human epidermoid carcinoma No. 2 cells and to L1210 cells. Administration of pyridoxal, which could saturate the endogenous aldehyde oxidase and thus delay the production of carboxyphosphamide, in combination with cyclophosphamide increases the life-span of mice implanted with L1210 cells. The metabolic conversion of nicotine to cotinine by liver proceeds in the same manner as cyclophosphamide oxidation. Nicotine is also oxidized by an amine oxidase to nicotine 1′-oxide. Lung homogenates accomplish the initial oxidation of both cyclophosphamide and nicotine but do not metabolize the products further. Kidney homogenates contain the amine oxidase producing nicotine 1′-oxide. Several other tissues are not active in the metabolism of either cyclophosphamide or nicotine.

Inhibition of DNA and RNA metabolism by daunorubicin and adriamycin in L1210 mouse leukemia.

Abstract: Summary Effects of daunorubicin and its new analog, adriamycin, on nucleic acid metabolism were studied in vitro in L1210 mouse leukemia cells with labeled nucleoside precursors and were compared with the established effects of actinomycin D. L1210 ascites tumor cells incubated with daunorubicin under physiological conditions of pH, temperature, and tonicity showed significantly greater inhibition of tritiated thymidine incorporation into DNA and 14 C-labeled uridine incorporation into RNA than cells treated with equimolar concentrations of adriamycin. Investigation of cell-drug interaction showed that uptake of daunorubicin by L1210 cells was substantially greater than adriamycin uptake at 37°; also, daunorubicin was metabolized to daunorubicinol whereas adriamycin did not undergo a similar conversion. Differences in cellular uptake of drug probably play a significant role in the inhibition of nucleic acid metabolism by daunorubicin and adriamycin in L1210 cells in vitro . The importance of other mechanisms operative in determining difference in overall therapeutic efficacy in vivo remains to be established.

Studies Correlating the Growth Rate of a Tumor and Its Metastases and Providing Evidence for Tumor-related Systemic Growth-retarding Factors

Abstract: Summary Previous studies have shown that the growth of a solid tumor is characterized by progressive slowing of the rate of growth as the tumor increases in size, but it is not clear to what extent local or systemic factors determine this slowing of growth rate. In this study, the growth of an implanted, isologous Lewis lung carcinoma in C57BL/6 mice was followed by serial tumor diameter measurements, and the number of tumor cells which had metastasized to lungs and kidneys was followed by serial quantitative transplant bioassay. Tumor diameter data were converted to tumor weight by a previously derived formula. Synchronous slowing of the rate of growth of the implanted tumor and its metastases was observed. Early after transplantation, the implanted tumor grew exponentially, and early lung metastases grew exponentially at a similar rate. Later, as the growth rate of the large implanted tumor slowed, a similar degree of slowing of the growth rate of metastases in lung and kidney was observed, even though these metastatic foci were microscopic in size. Host immunological factors did not seem to be involved, since challenge of animals at various stages of tumor growth with graded doses of tumor cells showed no evidence of transplantation resistance. Synchronous slowing of tumor growth rate was also demonstrated in experiments in which metastases were simulated by a 2nd implant. Following removal of the primary tumor, this slowing of tumor growth was reversible in both the lung metastases and the simulated metastases. These studies point to the importance of nonimmunological systemic factors (nutritional deficiencies or tumor by-products) that influence tumor growth rate.

Collagenolytic enzymes in human neoplasms.

Abstract: A variety of human neoplasms were examined for their ability to produce collagenolytic enzymes in culture. Some types of tumors of epithelial origin demonstrated a very high frequency of collagenolytic activity, while neoplasms of mesenchymal origin, nonneoplastic tissues, and other types of epithelial neoplasms only rarely produced collagenase. In particular, tumors of the colon and carcinomas of both squamous and basal cell origin displayed a high frequency of activity. Tumor collagenases from several different sources were isolated and examined for their mode of attack on native collagen, pH optima, and inhibition of activity by EDTA, cysteine, and pooled human serum. Tumor enzymes appear to be similar to the collagenases isolated from normal human skin by these criteria.

Persistent Binding of a New Reaction Product of the Carcinogen N-Hydroxy-N-2-Acetylaminofluorene with Guanine in Rat Liver DNA in Vivo

Abstract: The binding of 2-acetylaminofluorenyl residues (-AAF) to rat liver DNA in vivo was studied at different periods of time after administration of N -hydroxy- N -2-acetylaminofluorene-2′-3H. Liver DNA was hydrolyzed at pH 6 with a mixture of spleen phosphodiesterase and wheat germ acid phosphatase. The enzymatic digests were analyzed by Sephadex LH-20 column chromatography. Maximum levels of bound radioactivity were found in liver DNA at 16 to 18 hr following a single injection of N -hydroxy- N -2-acetylaminofluorene-2′-3H. The major part (80%) of the bound radioactivity was identified as N -(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF), which disappeared rapidly from DNA with a biological half-life of approximately 7 days. A second product, however, constituting 20% of the bound radioactivity, remained associated with DNA for periods of up to 8 weeks after injection. Unlike dGuo-AAF, the minor product was not deacetylated by the action of 0.1 n NaOH or 0.1 n HCl at 75° for 2 hr. The persistent -AAF residue was not detected in rRNA and tRNA from rat liver. A minor product with chemical and chromatographic properties similar to those of the persistent -AAF moiety in vivo was isolated from 14C-labeled DNA, which had been reacted with N -acetoxy- N -2-acetylaminofluorene-2′-3H in vitro . The 3H:14C ratio of this product was identical to the theoretical 3H:14C ratio, which was calculated for the reaction product dGuo-AAF, thus indicating that the persistent -AAF moiety in DNA is also bound to guanine. Persistent binding of radioactivity to rat liver DNA in vivo was also observed following a single injection of 2-acetylaminofluorene-9-14C-2′-3H. Previous reports that 70% of the total carcinogen bound to DNA at 24 hr had lost the N -acetyl group were confirmed. Approximately 20% of bound -AAF at 24 hr (5% of total carcinogen) remained associated with DNA at 4 weeks after injection. The 3H:14C ratio of DNA at 4 weeks was identical to the 3H:14C ratio of the isolated reaction product dGuo-AAF, indicating that there are no persistent 2-aminofluorene residues. Administration of 2-acetylaminofluorene-9-14C-2′-3H resulted in 53% exchange of N -acetyl groups, but injection of N -hydroxy- N -2-acetylaminofluorene-9-14C-2′-3H did not result in significant exchange of the N -acetyl group.

Combination Cancer Therapy: Presidential Address

Abstract: TheUniversityofTexas,MDAndersonHospitalandTumorinstitute,TexasMedicalCenter,Houston,Texas77025FirstIwanttothankyoufortheprivilegeandhonorofservingasyourPresidentduringthispastyearIthasbeenaturbulentandanimportantyearforcancerresearchandacceleratedsupportisinprocessIwouldliketotakethisopportunitytopresentsomeoftheconceptualandpracticaladvancesthathavebeenmadeincancerchemotherapyinthepast15to20yearsandtooutlinefutureprojectionsIn1955IarrivedattheNationalCancerInstituteTheneedforthedevelopmentofsystemictreatmentforcancer,theencouragementfrommysuperiors,andtheopportunitytoobservetheverydramaticeffectthatcouldbeachievedoccasionallyintheleukemiasthroughtreatmentdevelopedbyDrSFarberandDrJHBurchenalconfirmedmyinterestinmedicaloncologyandchemotherapyIhavebeenextraordinarily fortunateinthesuperiors,associates,andtraineeswithwhomIhavebeenassociatedIwouldliketomentionthreespecificallyWhenIarrivedattheNationalCancerInstitute,itwasDrGordonZubrodwhoprovided thefriendliness, stimulation, guidance, andopportunitytowork,andhehascontinuedtodosoFivemonthsaftermyarrival,agentlemanappearedinmyofficeandannouncedthathisnamewasEmilFreireichIallowedthat,asidefrombeingsomewhatlong,thiswasindeedaveryexcellentnameandthatanyonewithanamelikethathadto1Presentaddress:Children'sCancerResearchFoundation,Inc,35BinneyStreet,Boston,Mass02115ReceivedAugust23,1972;acceptedAugust23,1972begoodIofferedhimajobonthespot,whichwaseasilyoneofthesmartestthingsIeverdid;wehavebeenworking,playing,andfightingtogethereversinceLastbutnotleastIwantedtointroducemywifeElizabethUnfortunately,shecouldnotattendbecauseoftheurgencyofherworkonbehalfofattainingpeaceinIndo-ChinaSeventeenyearsagothechemotherapistwasisolatedfromhisfellowcliniciansinotherdisciplinesrelatingtocancermainlybecausehehadrelativelylittletoofferHewasalsoisolatedfromthebridgingandfromthebasicscientists,partiallybecausethefieldwasinitsveryearlystagesofdevelopmentbutlargelybecausemostscientists,clinicalandotherwise,werescepticalconcerningthefutureofcancerchemotherapyAschematicdiagramofdrugtreatmentcategoriesofdisseminatedcancerispresentedinChart1OurgoalistoridthepatientofhisneoplasticcellsUsingrathercrudetechniques,suchasChalklycounts,todeterminethenumberofneoplasticcellsatautopsy,wefoundthatpatientswithdisseminatedneoplasticdiseasehaveapproximately onetrillionorIO12cells(8)Similarvalueshavebeenobtainedviamoresophisticatedtechniquesinmyelomabydividingthetotalparaproteinproductionbytheamountofparaproteinproducedbyindividualcells(23)Theinitialcategoryiscalledremissioninductionwhereinthegoalistoproducecompleteremission,thatis,toeliminateallclinicalevidenceofthediseaseThisnecessitatesatleasta99%reductioninneoplasticcellsor,touse acolloquialism, 2-logorgreaterreductioninthenumberofneoplasticcellsOncethisisachieved,treatment duringremissionisinitiated Forrelativelyhomogeneouspopulationsoftumorcells,aIst-orderkineticeffectoftreatmentobtainsThismeansthatittakesjustasmuchtreatmenttoreducethenumberofneoplasticcellsfromamilliontoathousand(lessthan1mgoftissue)asitdoestoreducethenumberofneoplasticcellsfromIO12toIO9(1kgoftumor)Thus,theproductionofcompleteremissionrepresentsonlythedestructionofthetopofthe"exponentialiceberg,"andcontinuedtreatmentinremissionisnecessaryAsthenumberofcellsarereducedthereiscytokineticandmoredirectexperimentalevidencethattheslopeofthecurvewithtreatmentmayinfactbecomemoresteep,mainlybecausealargerproportionofthecellsentercycleThereisalsoexperimentalevidencethatnegotiatingthefinalhurdlefromalmostcompleteeradicationofneoplasticcellstocompleteeradicationmaynecessitateanalteredapproachtoDECEMBER1972 2593

The Proliferation of Capillary Endothelial Cells

Abstract: Recognition of capillary endothelial cells was facilitated by a special staining procedure developed previously. Thymidine-labeling indices for endothelial cells were estimated in a number of unstimulated mouse tissues and were found to be in the range 0 to 2.4%. There was no increase in mean labeling index up to 3 weeks after 2000 rads or up to 2 weeks after 4000 rads irradiation of muscle, skin, or bone. After fracture of the femur, the labeling index of capillary endothelial cells in regenerating callus increased to about 10% at 3 to 5 days after fracture and decreased to zero by Day 16. The duration of DNA synthesis in the fractured femur was estimated in a double-labeling experiment to be 7 ± 2 hr. The results of a repeated labeling experiment suggested a turnover time of 80 ± 25 hr on the 6th day after fracture, with a wide distribution of intermitotic times about the mean value. At 3 days after injury, when the labeling index is higher, the corresponding estimate of turnover time is about 50 hr: this value is close to a previous estimate of turnover time for capillary endothelial cells of a mammary tumor growing in the same strain of mouse.

Comparative Pharmacokinetics of Daunomycin and Adriamycin in Several Animal Species

Abstract: Summary The physiological disposition of adriamycin and daunomycin has been studied in several animal species. Both drugs were rapidly cleared from plasma, deposited in tissues, and then excreted slowly. Drug and metabolites were excreted via the bile and urine. Daunomycin was extensively metabolized by mice, rats, dogs, and hamsters, whereas no evidence was obtained for the metabolism of adriamycin in rats and mice. Hamsters metabolized adriamycin to an aglycone. The metabolite of daunomycin in all species appeared identical to daunorubicinol, described by Bachur and Gee. In hamsters, daunorubicinol as well as another metabolite of daunomycin were observed. This unknown metabolite, D x , appeared to be an aglycone but not the aglycone of daunomycin or daunorubicinol. The aglycone, D x , was also observed in several tissues of rats and mice that were exposed to hypobaric stress.

Potentiation of Drug Effect by Tween 80 in Chinese Hamster Cells Resistant to Actinomycin D and Daunomycin

Abstract: Summary In vitro studies of Chinese hamster sublines have indicated that cellular resistance to actinomycin D and daunomycin is predominantly due to decreased uptake of drug. Very probably, the alteration mediating resistance occurs in the external cell membrane. For substantiation of these previous findings, sensitive and resistant sublines were grown in the presence of graded concentrations of antibiotics, of the surface-active, nonionic detergent Tween 80 and of various combinations of both. Tween 80 caused a marked potentiation of drug effect, particularly in resistant cells, which exhibited a considerable degree of cross-resistance to the surfactant. Increased uptake of tritiated actinomycin D and daunomycin in the presence of Tween 80 was shown by radioautography to be directly related to concentration of detergent. Both potentiation of drug effect in growth studies and enhancement of uptake of antibiotics strongly support the concept that experimentally developed resistance to antibiotics in these Chinese hamster cells is in large part due to an acquired alteration manifest at the cellular membrane.

Influence of insulin deprivation on growth of the 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats subjected to alloxan diabetes and food restriction.

Abstract: Summary The present study investigates the possibility that insulin dependence, a property exhibited in organ culture by a majority of the 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinomas, might also be operating in vivo. It was found that induction of alloxan diabetes 3 or 4 weeks after 7,12-dimethylbenz(a)anthracene administration completely prevented mammary tumor formation. Moreover, induction of alloxan diabetes in tumor-bearing rats produced the rapid regression of 90% of the tumors, with a time course similar to that observed after oophorectomy or hypophysectomy. In contrast to what occurs after oophorectomy but in accordance with what is observed after hypophysectomy, administration of estradiol benzoate failed to prevent the tumor regression produced by alloxan diabetes. Because induction of alloxan diabetes involves a considerable loss of body weight, the effect on the mammary tumors of a severe food restriction, leading to an even greater loss of body weight, was also studied. It was found that food restriction resulted in a rapid regression partially counteracted by estradiol benzoate. Tumors regressing as a result of alloxan diabetes, those progressing (about 10%) in spite of diabetes, and those that progressed under the stimulating effect of estradiol benzoate in rats subjected to food restriction were investigated with respect to their insulin dependence in organ culture. It was observed that the tumors regressing in alloxan-diabetic rats were, by and large, markedly insulin dependent in vitro, whereas those growing despite diabetes were little- or noninsulin dependent. Eventually, most tumors that were stimulated to grow by estradiol benzoate in food-restricted rats proved very insulin dependent in vitro. These observations suggest that the tumors that are insulin dependent in organ culture similarly have a stringent requirement for insulin to grow in vivo and that they regress after induction of alloxan diabetes as a consequence of insulin deprivation. This would explain why only tumors that are insulin independent in vitro are able to grow despite the insulin deprivation of alloxan diabetes. It is conceivable that food restriction results in tumor regression also as a consequence of a decreased rate of insulin secretion. However, this decrease would be smaller than in the diabetic state, and the available insulin would be present in amounts large enough to allow insulin-dependent tumors to grow, provided other limiting factors such as estrogens or estrogen-stimulated hormones are restored.

Malignant transformation of cells derived from mouse prostate by epoxides and other derivatives of polycyclic hydrocarbons.

Abstract: Summary K-region epoxides of benz[a]anthracene, dibenz[a,h]anthracene, and 3-methylcholanthrene have been found to be toxic and more active in producing malignant transformation of cells derived from mouse prostate than their respective parent hydrocarbons and K-region dihydrodiols and phenols. The data support the view that metabolism of these polycyclic hydrocarbons is a prerequisite for their biological activity. 7-Bromomethylbenz[a]anthracene, the K-region epoxide of 7-methylbenz[a]anthracene, and 7-bromomethyl-12-methylbenz[a]anthracene were either inactive or less active in producing malignant transformation than the parent compounds, 7-methylbenz[a]anthracene and 7,12-dimethylbenz[a]anthracene. The 8,9-epoxide (non-K-region) of benz[a]anthracene was much less active than the K-region epoxide of this hydrocarbon; and the K-region epoxides of the noncarcinogenic hydrocarbons, phenanthrene and chrysene did not transform the cells.

Cell Cycle Phase Specificity of Antitumor Agents

Abstract: Summary The sensitivity to drugs of synchronous and asynchronous populations of DON cells was studied. Agents that were cytotoxic at a specific phase of the cell cycle gave dose-survival curves that decreased to a constant saturation value. DNA synthesis inhibitors such as 1-β-d-arabinofuranosylcytosine, 5-azacytidine, 5-hydroxy-2-formylpyridinethiosemicarbazone (NSC 107392), sodium camptothecin (NSC 100880), 5-fluorodeoxyuridine, and pseudourea (NSC 56054) were most cytotoxic to cells in the S phase. However, the DNA synthesis inhibitor, neocarzinostatin, was most cytotoxic to cells in the G 1 phase. The protein synthesis inhibitors, pactamycin and sparsomycin, were also most cytotoxic to cells in the S phase. Cells in the G 1 -S border region were most sensitive to the RNA synthesis inhibitors, actinomycin D and nogalamycin, and to the alkylating agents, 1,3-bis(2-chloroethyl)-1-nitrosourea, and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. Streptozotocin and tubercidin, which markedly inhibit the synthesis of DNA, RNA, and protein, were cytotoxic to cells in all phase of the cell cycle. 5-Fluorouracil was also not phase specific. Cells in mitosis and in the G 1 phase were most sensitive to chlorambucil, l-phenylalanine mustard, and ellipticine.

On the sulfate ester of N-hydroxy-N-2-fluorenylacetamide as a key ultimate hepatocarcinogen in the rat.

Abstract: Summary The problem of the biochemical activation steps required to elicit carcinogenicity to rat liver by N -2-fluorenylacetamide was investigated by bioassay, competitive inhibition, and biochemical techniques. Acetanilide inhibits not only the hepatocarcinogenicity of N -2-fluorenylacetamide but also that of the carcinogenic N -hydroxylated metabolite. With the parent compound, the inhibition was traced chiefly to competition at the N -hydroxylation step, which should not apply to the N -hydroxy derivative. With the latter, the addition of sodium sulfate to the diet restores the hepatocarcinogenicity of N -hydroxy- N -2-fluorenylacetamide in the presence of inhibitory acetanilide. However, sulfate addition fails to return carcinogenicity in the system N -2-fluorenylacetamide plus acetanilide, wherein the inhibition lies at the N -hydroxylation step. The combined results provide evidence that sulfate ester formation constitutes a required second activation step for the expression of carcinogenicity to the liver of rats of N -hydroxy- N -2-fluorenylacetamide, obtained by a first activation step. The required sulfotransferase enzyme system is found mainly in liver cytosol, but attempts to locate it in rat liver nuclear fractions resulted in the detection of only questionable amounts.

The role of lymphatic obstruction in the formation of ascites in a murine ovarian carcinoma.

Abstract: Summary In normal mice, 60 to 70% of 51Cr-labeled erythrocytes injected i.p. appear in the peripheral bloodstream within 5 hr. In mice previously inoculated with a transplantable, ascites-producing ovarian tumor, this egress of labeled erythrocytes from the abdominal cavity is significantly impaired before ascites develops. Diaphragmatic lymphatic obstruction is demonstrated histologically. Since red cells leave the peritoneal cavity primarily by way of these lymphatic channels, these observations suggest that lymphatic obstruction by tumor cells is probably of pathogenetic importance in the accumulation of ascitic fluid.

The Inhibition of Croton Oil-promoted Mouse Skin Tumorigenesis by Steroid Hormones

Abstract: Tumors in female Swiss Millerton mouse skin were initiated with 25 µg 7,12-dimethylbenz(a)anthracene and, 2 weeks later, were promoted 3 times/week with 0.2 ml of 0.5% croton oil. Four steroid hormones in 0.2 ml of acetone were applied percutaneously at 6 and 30 µg 5 times/week. A fifth steroid was applied at the 30-eg dose. Tumor inhibition by the hormones was dose dependent in the order dexamethasone > Schering No. 11572 > prednisolone > hydrocortisone > cortisone. There was a general correlation between the antiinflammatory and antitumor activities of the steroids.

Coordinated Treatment of Childhood Rhabdomyosarcoma with Surgery, Radiotherapy, and Combination Chemotherapy

Abstract: Summary Twenty children with rhabdomyosarcoma were treated in a coordinated program utilizing surgery, radiotherapy, and combination chemotherapy. Therapy was determined by the stage of disease at which treatment was initiated. Of the 20 patients, 15 developed complete regression of tumor; 4 patients developed partial response. Of the 9 patients who are alive, 7 have been tumor free for 2 to 39 months. Concurrent radiotherapy and prolonged, intensive, combination chemotherapy are well tolerated following surgery for rhabdomyosarcoma. The coordinated use of these modalities offers prospects of curing more children who have this tumor.

Inhibition of Tumorigenesis in Mouse Skin by Leupeptin, a Protease Inhibitor from Actinomycetes

Abstract: Leupeptin, with the structure N -acetyl( N -propionyl)-l-leucyl-l-leucyl-dl-argininal, was isolated from Actinomycetes and is a potent and specific inhibitor of proteases. In this work, leupeptin was found to inhibit tumorigenesis in mouse skin induced by a single, noncarcinogenic dose of 7,12-dimethylbenz(a)anthracene followed by repeated application of croton oil. Tumors that had already been induced were scarcely affected by leupeptin. The activity of p -toluene-sulfonyl-l-arginine methyl ester esterase in mouse skin was markedly increased by treatment with croton oil and was inhibited by leupeptin both in vitro and in vivo . The effect of leupeptin in repressing tumorigenesis seems to be due to its inhibition of p -toluene-sulfonyl-l-arginine methyl ester esterase in the skin and not to a direct effect on tumors.

Detection of avian and mammalian oncogenic RNA viruses (oncornaviruses) by immunofluorescence.

Abstract: Summary The indirect immunofluorescence and immunofluorescence absorption tests were used to determine the presence and intracellular deposition of viral antigens in cells infected with avian, murine, hamster, and feline oncornaviruses In all instances, viral antigens were restricted to the cytoplasm; nuclear fluorescence was not observed Screening of murine neoplasms by immunofluorescence and immunofluorescence absorption showed ( a ) that murine leukemia virus and murine mammary tumor virus (MTV) were serologically unrelated; ( b ) that murine leukemia virus was widespread throughout the mouse population and occurred in a variety of tumors and leukemias of mice from both high- and low-leukemia-incidence strains; and ( c ) that MTV was restricted to a few types of neoplastic tissues, which occurred only in mice of high-mammary-tumor-incidence strains With the immunofluorescence absorption technique, MTV, as well as murine leukemia virus, could be detected in the spleen of individual mice from high-incidence strains The amount of viral antigen in the spleen of mice from crosses of high-mammary-tumor strains (or high-leukemia strains) with mice of low-incidence strains was intermediate between that of the parental strains A serological comparison of murine oncornaviruses with those of the chicken, hamster, and cat revealed that ( a ) the avian and mammalian oncornaviruses are serologically unrelated to each other; ( b ) oncornaviruses from the chicken, mouse, hamster, and cat contain species-specific viral antigens that are serologically distinguishable (however, the leukemia and sarcoma viruses within a single species are antigenically indistinguishable); ( c ) the mammalian leukemia-sarcoma viruses share at least one common antigen; and ( d ) the mouse MTV is serologically unrelated to all other oncornaviruses

Metabolism and Biochemistry of Mycophenolic Acid

Abstract: Summary Mycophenolic acid is a new, experimental oncolytic agent that interferes in the interconversion of inosine, xanthosine, and guanosine monophosphates. IMP dehydrogenase, which converts IMP → XMP, and GMP synthetase, which converts XMP → GMP, are inhibited by mycophenolic acid. The IMP dehydrogenase from a human adenocarcinoma of the colon was more sensitive to mycophenolic acid than was the enzyme from murine tumors. There is a correlation between the sensitivity of experimental tumors to mycophenolic acid and the relative activities of β-glucuronidase and hypoxanthine-guanine phosphoribosyltransferase. Mycophenolic acid glucuronide is unable to cross the cell membrane; therefore the intracellular concentration of the free acid depends on the rate of hydrolysis of the glucuronide by β-glucuronidase. A mechanism of resistance to mycophenolic acid is the circumvention of the block in the nucleotide interconversion. GMP is resupplied by conversion of guanine to its nucleotide by hypoxanthine-guanine phosphoribosyltransferase. In tumors resistant to mycophenolic acid, the transferase activity is high; in tumors sensitive to mycophenolic acid, the activity is low. The relative activities of these two enzymes in tumors in man may indicate the potential effectiveness of mycophenolic acid in humans. Mycophenolic acid is initially secreted into the bile and excreted in the urine and feces of animals. 14CO2 was not detected in the expired air of mice, rats, or marmosets given 14C-labeled mycophenolic acid. The only metabolite detected in mice, rats, rabbits, and humans was mycophenolic acid glucuronide.

Imbalance in ornithine metabolism in hepatomas of different growth rates as expressed in formation of putrescine, spermidine, and spermine.

Abstract: Summary In comparison with normal livers from rats of the same age, sex, and dietary regimen, the l-ornithine decarboxylase (EC 4.1.1.17) activities of fast-growing Morris hepatomas (e.g., lines 3924A and 7777) were very high, whereas a number of slow-growing and more differentiated hepatomas exhibited l-ornithine decarboxylase activities which were considerably less elevated. None of the hepatomas examined, regardless of their speed of growth, had significantly elevated S-adenosylmethionine decarboxylase activities in comparison with that of liver. The steady-state concentration of putrescine in some but not all of the fast-growing neoplasms was much greater than that in the corresponding normal liver controls. The putrescine content of all slow-growing hepatomas was usually above normal hepatic range. The spermidine/spermine ratio tended to be higher in all of the hepatomas studied than in the corresponding normal livers; the overall concentrations of these two polyamines in all tumors were, however, within the range seen in the normal livers (0.6 to 1.5 µmoles/g.) The utilization of l-ornithine for aliphatic polyamine synthesis versus the operation of the urea cycle in rat liver and its neoplasms is discussed.

Specific Estrogen-binding Capacity of the Cytoplasmic Receptor in Normal and Neoplastic Breast Tissues of Humans

Abstract: Carcinomas and nonmalignant breast tissues from 89 women undergoing surgery were examined for the presence of specific estrogen-binding substances by a radioligand binding technique in vitro . Following incubation of the 105,000 × g supernatant fraction with estradiol-17β-6,7-3H of high specific radioactivity, in the presence or absence of a known antiestrogen, specific receptors for estra-1,3,5,(10)-triene-3,17β-diol-3H were identified by isotopic profiles from sucrose gradient analyses. Cytoplasmic estrogen receptors from breast carcinoma migrated with a sedimentation velocity coefficient of approximately 8 to 9 S. Cytosols from 29 primary carcinomas were classified as positive, exhibiting an average binding capacity of 43.0 ± 5.3 fmoles/mg protein, with a range of 10.3 to 137.6 fmoles/mg protein. An additional 10 carcinomas were considered borderline on the basis of an average binding capacity of 5.6 ± 0.4 fmoles/mg protein, with a range of 3.3 to 7.4 fmoles/mg protein. Thirty-six of 75 tumors displayed insignificant specific estrogen binding. Inhibition of hormone binding by the competitive inhibitor, CN-55,945-27, averaged 87 ± 3% for the tumor cytosols exhibiting elevated binding; specific binding by cytosols with low receptor capacity was inhibited by 81 ± 5%. In addition to these neoplasms, 16 specimens of normal breast, 5 of fibrocystic disease, and 1 of fibroadenoma were analyzed; all but one of these exhibited insignificant specific binding. These data confirm the presence of specific estrogen-binding substances in certain infiltrating ductal carcinomas of the human breast. Twenty-three of the 29 carcinomas exhibiting specific estra-1,3,5,(10)-triene-3,17β-diol-binding capacity were from postmenopausal women 55 years of age or older. Specific estra-1,3,5,(10)-triene-3,17β-diol-binding capacity of a tumor was apparently not related to the presence of metastases, nor was it related to the estimated percentage of carcinoma cells in the specimen examined.

Interaction of 1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea (NSC 79037) with Nucleic Acids and Proteins in Vivo and in Vitro

Abstract: The macromolecular binding of radioactivity from 1-(2-chlorethyl)-3-cyclohexyl-1-nitrosourea, labeled with 14C in either the cyclohexyl moiety or the ethylene residue, was studied in the L1210 leukemia-bearing mice, in a suspension of L1210 leukemia cells, and during in vitro incubation with isolated nucleic acids and proteins. In all three systems, radioactivity from cyclohexyl-14C-labeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was extensively bound to proteins, and there was negligible binding to nucleic acids. Radioactivity from the ethylene-14C-labeled drug was bound to both nucleic acids and proteins, but the binding was only a fraction of the observed protein binding of the cyclohexyl label. Of the various macromolecules examined for in vitro binding, poly-l-lysine and albumin were the most active in binding the cyclohexyl-labeled material; polyguanylic acid, polycytidylic acid, and tRNA were the most active in binding the ethylene-14C-labeled material. The possibility that the carcinostatic activity of this drug reflects a dual capacity, namely, modification of cellular proteins via cyclohexylcarbamoylation and of nucleic acids via alkylation, is discussed.

Respiratory Tract Carcinogenesis in Hamsters Induced by Different Numbers of Administrations of Benzo(a)pyrene and Ferric Oxide

Abstract: Benzo(a)pyrene (BP), prepared as a suspension of fine crystalline particles attached to particles of ferric oxide in 0.9% NaCl solution, was administered by intratracheal instillation to Syrian golden hamsters. A single administration of 37.5 mg BP with 12.5 mg ferric oxide in 0.5 ml 0.9% NaCl solution induced five bronchogenic carcinomas and five histologically benign respiratory tumors in a total of 61 hamsters. A single administration of 5 mg BP with 45 mg ferric oxide in 0.5 ml 0.9% NaCl solution induced one peripheral adenocarcinoma and six histologically benign respiratory tumors in a total of 189 hamsters. Repeated instillations, each of 3 mg BP with 3 mg ferric oxide in 0.20 ml 0.9% NaCl solution, were administered 5, 10, or 15 times; two experimental groups were used at each dose level. Respiratory tract tumors, mostly bronchogenic carcinomas, were induced in all groups, and a positive dose-response relationship was demonstrated.

Therapeutic Efficacy of Cyclophosphamide as a Function of Its Metabolism

Abstract: Experiments were designed to investigate the metabolism of cyclophosphamide in vitro and in vivo following the administration of known stimulators and depressors of rat hepatic microsomal mixed-function oxidase activity, the antitumor efficacy of cyclophosphamide as a function of its metabolism, and the toxicity of cyclophosphamide as a function of its metabolism. Regarding the metabolism of cyclophosphamide by hepatic microsomal preparations, the following observations were made. ( a ) Pretreatment with Phenobarbital of male and female rats and of female mice increased the rate of metabolism 7-, 23-, and 7-fold, respectively. ( b ) pretreatment of male rats with 3-methylcholanthrene depressed metabolism to 33% of that of controls, but similar pretreatment of female mice did not alter the rate of metabolism. ( c ) The Km for cyclophosphamide metabolism by microsomes obtained from male rat liver changed from 1.39 mm to 0.57 and 0.58 mm after phenobarbital and 3-methylcholanthrene pretreatment, respectively. ( d ) Pretreatment of male rats with thioacetamide, morphine, or cobalt chloride depressed metabolism to 12, 48, and 8% of control, respectively. ( e ) Male rats bearing the Walker 256 carcinosarcoma i.m. showed a depressed ability to metabolize cyclophosphamide. In vivo cyclophosphamide metabolism in rats paralleled in vitro metabolism. Thus, at early time points, blood levels of alkylating activity were ( a ) increased following pretreatment with phenobarbital, ( b ) decreased following pretreatment with 3-methylcholanthrene or cobalt chloride, ( c ) decreased when the animal bore the Walker 256 carcinosarcoma i.m., and ( d ) lower in female compared with male rats. Walker 256 carcinosarcoma cells grown i.m. in the hindlegs of male and female rats were used to evaluate therapeutic efficacy. The dose of cyclophosphamide that inhibits tumor growth in male rats by 50% was 0.7 mg/kg. Similar median effective doses were obtained in female rats and in male rats pretreated with phenobarbital or cobalt chloride. 3-Methylcholanthrene pretreatment increased the median effective dose of cyclophophamide to 3.5 mg/kg. For estimation of the toxicity of cyclophosphamide, blood leukocyte counts were made at various intervals following injection of cyclophosphamide. Little difference in the decline of the number of leukocytes or in their subsequent return to normal levels was observed between control male rats; female rats; and phenobarbital-, 3-methylcholanthrene-, or cobalt chloride-pretreated male rats. Pretreatment with phenobarbital did accelerate leukocyte depression and also increased the magnitude of depression. In methylcholanthrene-pretreated male rats and in female rats, the magnitude of depression was somewhat less and recovery rates were somewhat altered. Administered by itself, phenobarbital, 3-methylcholanthrene, or cobalt chloride had no effect on tumor growth or blood leukocyte levels. The data demonstrate the futility of trying to improve the therapeutic efficacy of cyclophosphamide by pretreatment with drugs that alter its rate of activation. In addition, the data provide a rational basis for the ineffectiveness of such an effort.