Showing papers in "Cancer Research in 1974"

Quantitative relationships of intravascular tumor cells, tumor vessels and pulmonary metastases following tumor implantation.

Abstract: Summary An experimental model has been developed to quantify some of the major processes initiated by tumor transplantation and culminating in pulmonary metastases. The T241 fibrosarcoma, chosen because of its high hematogenous metastatic propensity and reproducible biological behavior, is transplanted into the femoral region of the C57BL mouse. Experiments are performed at specified times after transplantation to determine the dynamics of tumor growth, density, and size distribution of perfused tumor vessels, entry rate of tumor cells into the circulation, and the number of pulmonary metastases. Estimates of the entry rate of tumor cells into the circulation are obtained by perfusing tumors with an oxygenated, cell-free medium to allow the counting of single tumor cells and tumor cell clumps collected in the venous effluent. The tumor vascular network appears at approximately Day 4, while tumor cells are first observed in the perfusate approximately 5 days after transplantation. The concentration of effluent tumor cells, singly and in clumps, increases rapidly until Day 10, after which the rate of cell entry into the perfusion is diminished. A linear relation is found between the density of perfused vessels and the concentration of effluent tumor cells. Metastases, first observed on Day 10, increase rapidly with time, at a rate similar to that of the concentration of effluent tumor cell clumps containing 4 or more cells. These studies suggest that dynamics of hematogenously initiated metastases depend strongly on the entry rate of tumor cell clumps into the circulation, which in turn is intimately linked to tumor vascularization.

Errors in DNA Replication as a Basis of Malignant Changes

Abstract: Summary In this essay we have considered possible relationships between errors in DNA replication and malignant progression. Theoretical and experimental studies designed to determine the energy of interaction between different nucleotides have been reviewed. The difference of the energy of interaction between complementary and noncomplementary base pairs (1 to 3 kcal) is not sufficient to account for the exactness of DNA replication that is observed in vivo or in vitro. Thus, DNA polymerases must play a role in base selection. This point has been established by the detection of polymerases that copy polynucleotide templates with few errors and the detection of others that make many errors. Different mechanisms for base selection by polymerases have been considered. Our findings that both extracts of human leukemic lymphocytes and a purified DNA polymerase from avian myeloblastosis virus have exceptionally high error rates have been considered in relationship to oncogenesis. A hypothesis is presented that relates mistakes in DNA replication, as promoted by error-prone DNA polymerases, to tumor progression. We have also considered a more general concept encompassing early cellular changes that lead to oncogenesis. The justification for this essay is that both of these hypotheses are open to immediate experimental analysis.

Proceedings: Tumor angiogenesis factor.

Abstract: Summary Recent evidence from our studies indicates that solid tumor growth is not continuous but that it can be separated into two stages, avascular and vascular. In the avascular stage, tumors remain dormant at diameters of 1 to 2 mm. Further growth is possible only after new capillaries have been elicited from the host and have penetrated the tumor. This capillary proliferation is stimulated by a diffusible factor, tumor angiogenesis factor, released by solid tumors, and by neoplastic cells in culture. The biology and isolation of tumor angiogenesis factor are discussed. Evidence for the mechanism of tumor dormancy in the absence of angiogenesis will be presented. Therapeutic implications of the inhibition of tumor angiogenesis will be mentioned briefly.

Two Forms of Repair in the DNA of Human Cells Damaged by Chemical Carcinogens and Mutagens

Abstract: Summary DNA repair in human cells, as assayed by the photolysis of 5-bromodeoxyuridine incorporated during repair, takes one of two limiting forms, depending on the nature of the original insult to the DNA. Damage from ionizing radiation is repaired by the insertion of three or four nucleotides during a brief period (∼60 min) after the insult, but in the case of ultraviolet radiation there is extensive excision of bases (∼100) during a protracted period (18 to 20 hr). Similarly, chemicals that damage DNA fall into two categories, those that result in the ionizing or “short” type of DNA repair (ethyl methanesulfonate, methyl methanesulfonate, propane sultone) and those that result in the ultraviolet or “long” type of repair (N-acetoxy-2-acetylaminofluorene, 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino] acridine dihydrochloride). In addition, some chemicals (4-nitroquinoline 1-oxide) cause damage that apparently is repaired by both mechanisms. When agents that induce long repair in normal cells are used to treat cells from patients with the genetic disease xeroderma pigmentosum (characterized by extreme sensitivity to ultraviolet radiation), defective repair is seen. Thus it is possible that long repair involves the action of an endonuclease (thought to be lacking in xeroderma cells) on a distortion or intercalation in the DNA. Short repair may involve the simple excision and replacement of a few bases at the site of a single-strand break.

Diffusion and Convection in Normal and Neoplastic Tissues

Abstract: Summary The hydraulic conductivity of s.c. and hepatocarcinoma tissue was determined by in vivo and in vitro experiments. A single parameter was developed to calculate the ratio of diffusive to convective flux of solutes in the extravascular space. Tissue glycosaminoglycan content and solute molecular weight are the two most important parameters which may determine whether the extravascular transport of solutes occurs predominantly by diffusion or by convection. The analysis has implications for transport of metabolic nutrients and wastes, drugs, hormones, plasma proteins, and viruses in the extravascular space.

Metabolic aspects of the role of hyperthermia im mammalian cell inactivation and their possible relevance to cancer treatment.

Abstract: Summary The importance of the metabolic state of Chinese hamster cells (HA1) in relation to the sensitivity of the cells to supranormal heat shock (43°) was investigated, and the following results were noted. 1.The sensitivity of cells to hyperthermia (as well as their ability to repair heat-induced damage after 43°) is strongly related to their nutritional history. Chinese hamster cells chronically deprived of serum (and probably other medium components) become extremely heat sensitive. 2.The role of oxygen and glucose may be less important; chronic or acute deprivation of oxygen has only minor influence on survival of exponentially growing or plateauphase cells. Similar conclusions appear to be true for variations in available glucose concentrations. 3.An appreciable fraction of cells killed by heat in the absence of serum lyse either during or shortly after heat exposure. The presence of serum inhibits the lysis process. 4.At temperatures of 41–43°, cells are sensitized to the chemotherapeutic agents, nitrogen mustard and bleomycin, but not to a nitrosourea. This sensitization may be related to repair inhibition at the elevated temperatures. The importance of these findings in the possible utilization of hyperthermia as an adjunct to conventional therapy is discussed.

A biochemical mechanism for the delayed cytotoxic reaction of 6-mercaptopurine.

Abstract: 6-Thioguanosine-35S and β-2′-deoxythioguanosine-35S were identified in venom hydrolysates of RNA and DNA isolated from 6-mercaptopurine (MP)-35S-treated L5178Y cell cultures. Digestion with purified phosphodiesterases demonstrated that MP is incorporated as thioguanine (TG) nucleosides in 3′,5′-phosphodiester linkages in DNA and RNA. 3′-Nucleotides of TG-35S were released from 35S-labeled DNA and RNA by digestion with micrococcal nuclease plus spleen phosphodiesterase. The 5′-ribonucleotide of TG-35S was released from 35S-labeled RNA by digestion with venom phosphodiesterase, and the 5′-deoxyribonucleotide of TG-35S was released from 35S-labeled DNA by digestion with pancreatic deoxyribonuclease plus venom phosphodiesterase. A relationship between delayed cytotoxicity and incorporation of MP as TG into nucleic acids was apparent in the following observations. Mycophenolic acid protected L5178Y cells against the delayed cytotoxic effect of MP and suppressed MP incorporation into nucleic acids. A spontaneous development of partial tolerance to MP in two lines of L5178Y cells was associated with a reduced capacity for incorporation of MP as TG into DNA and RNA. 6-Methylthioinosine potentiated cytotoxic effects of MP in a partially tolerant cell line and stimulated incorporation of MP into DNA and RNA. Nucleic acid-incorporated TG was the major thiopurine derivative in MP-sensitive cells at the time of the delayed cytotoxic reaction to MP exposure. Pulse exposure of cells to equitoxic concentrations of MP-35S and TG-35S resulted in similar levels of incorporation of TG-35S in nucleic acids. The delayed cytotoxic activity of MP is probably a consequence of MP anabolism to TG nucleotides followed by incorporation of the latter into DNA. It is likely that the same biochemical mechanism is responsible for delayed cytotoxic effects of both MP and TG.

Prognostic Correlations and Response to Treatment in Advanced Metastatic Malignant Melanoma

Abstract: Summary Four-hundred twenty-six patients with disseminated melanoma were analyzed, all of whom were treated with chemotherapy. There was a high correlation with hepatic metastases and elevated lactic dehydrogenase levels. Although there were frequent false-positive lactic dehydrogenase levels, normal lactic dehydrogenase levels were consistently associated with a normal liver. Central nervous system disease was a major cause of morbidity and mortality in metastatic melanoma. Eleven % of the patients in this study had evidence of central nervous system disease at onset. The only treatment that was proven to be effective in these patients was whole-brain radiotherapy with concomitant dexamethasone administration. There were certain areas of anatomical involvement that correlated with an improved survival. These were patients that had pulmonary metastases only (median survival, 10 months) and patients with disease limited to the skin and s.c. tissue (median survival), 11 months). The median survival for all patients was 4.7 months. The response rate to a variety of treatment regimens with 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide was 18% (75 of 426) for all patients and 23% for evaluable patients only. There was an increased response to 5-(3,3-dimethyl-1-triazeno)imidiazole-4-carboxamide for nonvisceral involvement (28%), compared with visceral involvement (13%). The addition of 1,3-bis[2-chloroethyl-1-nitrosourea appeared to have an increased response rate for pulmonary and other visceral involvement, compared with 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide alone.

Photoscan Localization of GW-39 Tumors in Hamsters Using Radiolabeled Anticarcinoembryonic Antigen Immunoglobulin G

Abstract: Summary Photoscans and organ radioactivity were assessed in hamsters bearing cheek pouch grafts of a carcinoembryonic antigen (CEA)-producing human colonic carcinoma, GW-39; a human sarcoma, H.S.1; and a hamster amelanotic melanoma, A.Mel.3. The animals were given injections of 10 to 50 μCi 125I-labeled heterospecific anti-CEA immunoglobulin G (IgG) or normal IgG. The photoscans showed an increased uptake of radioactivity over the tumors and frequently over the areas of the thorax and urinary bladder, regardless of the tumor or the radiolabeled IgG preparation used. Even normal hamsters receiving either radiolabeled preparation showed an increased accretion of radioactivity over the thoracic region. A specific tumor localization, however, was demonstrated in animals bearing small ( The radioactivity recovered from GW-39 tumors borne in hamsters given injections of radiolabeled anti-CEA IgG revealed an increase of 7.5 to 20 times that recovered from other tissues. A slightly increased uptake of either the specific or nonspecific radiolabeled preparation was seen in the other, non-CEA-producing tumors studied, which was sufficiently increased over background radiation to permit visualization of the tumors by photoscanning, especially when the neoplasms were large and vascular. Evidently radiolabeled nonantibody components of heterospecific IgG can be localized in certain tumors and normal tissues by photoscanning. Nevertheless, tumor localization due to an antigen-antibody reaction, as we have found in CEA-producing GW-39 tumors treated with an anti-CEA antibody preparation, permits photoscan visualization of tumors too small to be demonstrated by radiolabeled normal IgG. It thus appears that CEA is a suitable tumor target for radioantibody by photoscanning.

Inhibition of pulmonary metastasis by intravenous injection of specifically activated macrophages

Abstract: Summary The ability of syngeneic macrophages from C57BL/6 mice bearing a progressively growing B16 melanoma to inhibit established pulmonary metastases in vivo was studied. Macrophages were cultured in vitro with supernatants obtained from cultures of B16 tumor cells alone or tumor cells and either normal, nonspecifically sensitized or specifically sensitized xenogeneic (rat) lymphocytes. The different groups of in vitro -treated macrophages were injected i.v. or i.p. into other C57BL/6 mice that had been given i.v. injections of 10,000 viable B16 melanoma cells 48 hr previously. The data demonstrated that specifically in vitro -treated macrophages injected i.v. but not i.p. into mice significantly reduced their number of established pulmonary metastases. Moreover, it appeared that the in vivo inhibition of tumor nodules was continuing at the time of sacrifice. These results support the experimental data that cytotoxic macrophages may occupy an important role in the defense against neoplasia. Furthermore, the application of xenogeneic activation of macrophages from tumor-bearing animals rendering them cytotoxic may provide a possible approach to therapy.

High-pressure liquid chromatographic analysis of benzo(alpha)pyrene metabolism and covalent binding and the mechanism of action of 7,8-benzoflavone and 1,2-epoxy-3,3,3-trichloropropane.

Abstract: Summary High-pressure liquid chromatography is a rapid and efficient method for the separation of benzo(a)pyrene metabolites. This new technique readily separates eight benzo(a)pyrene metabolites from the parent hydrocarbon. These are three dihydrodiols (9,10-, 7,8-, and 4,5-dihydrodihydroxybenzo(a)pyrene), three quinones (benzo(a)pyrene-1,6-dione, -3,6-dione, and -6,12-dione) and two phenols (9- and 3-hydroxybenzo(a)pyrene). This method was applied to the study of the mechanism of inhibitor action on the microsomal benzo(a)pyrene metabolism. 7,8-Benzoflavone, an inhibitor of aryl hydrocarbon hydroxylase, inhibits the covalent binding of benzo(a)pyrene to DNA and inhibits the formation of each of the metabolites. The lack of selective inhibition suggests that the 7,8-benzoflavone acts on the oxidase or a prior component of the microsomal electron chain. 1,2-Epoxy-3,3,3-trichloropropane inhibits benzo(a)pyrene disappearance, which also suggests an inhibitory effect on oxidase activity. This compound, however, also stimulates formation of benzo(a)pyrene binding to DNA, reduces the ratio of 3-hydroxybenzo(a)pyrene to 9-hydroxybenzo(a)pyrene formation, and completely eliminates the formation of the three dihydrodiols. This selective effect suggests a specific inhibition of hydrase activity with a lesser effect on oxidase function.

Immunological Resistance to Pulmonary Metastases in C3Hf/Bu Mice Bearing Syngeneic Fibrosarcoma of Different Sizes

Abstract: Summary Immunological resistance to pulmonary metastases of a syngeneic methylcholanthrene-induced fibrosarcoma (FSA) was studied in C3Hf/Bu mice that were actively immunized against or that had borne that tumor for varying times. Tumor metastases were induced by i.v. inoculation of FSA cells, and their number was determined 14 to 17 days later. The number of metastases was greatly reduced in mice that previously had been immunized against the same tumor but not in those immunized against syngeneic mammary carcinoma. This reduction was mediated through the activity of immune spleen and lymph node cells and was related to the number of immune cells transferred into the recipient mice. Spleen cells were found to be more effective than lymph node cells. The presence of the s.c.-growing tumor reduced the number of pulmonary metastases. This was noted when s.c. implantation of FSA preceded the i.v. inoculation of the tumor cells by 3 hr and 3, 7, and particularly 14 days. Spleen cells from mice bearing s.c.-growing FSA for 3, 8, 12, 20, and 27 days were able to transfer immunity to pulmonary metastases of FSA in sublethally irradiated syngeneic recipients. Immunity was transferred most effectively by spleen cells from mice bearing the tumor for 12 days; this activity of the spleen cells markedly decreased in mice bearing the tumor for 27 days. Lymph node cells, however, were able to transfer antitumor immunity only if taken from mice bearing the tumors for 12 or 20 days. Serum from mice bearing FSA given i.v. to the tumor cell recipient mice as a single injection or in multiple doses did not prevent the reduction of pulmonary metastases by immune spleen and lymph node cells.

Selective Chemotherapy of Noncycling Cells in an in Vitro Tumor Model

Abstract: Summary The electron-affinic drug, metronidazole, preferentially killed noncycling cells in multicellular spheroids as determined using histology-autoradiography and colony-formation methods. This and other similar chemicals may be cytotoxic to the noncycling cells, due to their specific metabolic state and local environmental conditions, and should be useful in combination with cell cycle-specific agents in chemotherapy.

Cell cycle dependency of oncogenic transformation induced by N-methyl-N'-nitro-N-nitrosoquanidine in culture.

Abstract: Summary Malignant transformation has been induced by N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) in synchronized cultures of the C3H/10T1/2 CL8 line of mouse fibroblasts. This transformation shows marked cell cycle dependency, with the sensitive phase for malignant transformation located somewhere between 4 hr prior to S and the G 1 -S boundary. Cells were synchronized by arginine or isoleucine deprivation for 48 hr, by 2 mm thymidine for 24 hr, or by release from postconfluence inhibition of cell division. Replicate cultures were treated with MNNG, 4 µg/ml, at various times prior to, at, or after release of the block. DNA synthesis began 4 hr after release of cells from arginine or isoleucine deprivation, and about 90% of the cells doubled after 10 to 14 hr. A peak in transformation frequency (TF) occurred at the time of release in arginine-deprived cells, and 4 hr after release of the block in isoleucine-deprived cells. Synchrony induced by 2 mm thymidine was poorly defined; DNA synthesis began within 2 hr and cell division began within 4 hr after removal of the thymidine. The peak in TF occurred at the time of release of the block. When cells were released from postconfluence inhibition of cell division, a peak of TF was observed 4 hr prior to S phase, and a second peak of TF was located shortly before the second round of DNA synthesis. MNNG killed 99.9% of cells at the time of maximal TF, and toxicity increased as cells entered S phase. Arginine deprivation did not cause an alteration in chromosome number. Morphologically transformed colonies from MNNG-treated dishes were cloned, cultured, and injected into X-irradiated syngeneic mice. Nine of 10 transformed lines gave sarcomas, but the untreated line did not.

Epidemiological analysis of the most prevalent sites and types of canine neoplasia observed in a veterinary hospital.

Abstract: Summary A study of the epidemiology of canine neoplasia was conducted at the University of Pennsylvania School of Veterinary Medicine with data obtained from clinical and postmortem records for the period January 1, 1952, to December 31, 1963. Age-, sex-, and breed-specific ratios were obtained for the most popular breeds and the most prevalent types and sites. The overall prevalence of neoplasia in this population for the 12-year period was 4.2%. The highest relative risk was noted among boxers. This was true for a variety of types and sites of tumors. Age-adjusted data revealed a higher risk for boxers than nonboxers, particularly up to the age of 8 years. Again this was true for a variety of types and sites. Boxers were shown to have an unusually high prevalence of mastocytomas of the skin compared to nonboxers, but fewer mammary and circumanal tumors than expected. They also showed a particularly high relative risk of osteosarcomas and lymphosarcomas involving bone and lymph node as well as high relative risk for neoplasms of the testes and gingiva. An unusual distribution of age-specific ratios for lymphosarcomas and osteosarcomas was observed. Both presented similar age-specific ratios with a marked peak occurring between 7 and 10 years of age, followed by a marked decline. This was in contrast to other types or sites, which showed a continued increase in age-specific ratios with increased age.

Estrogen receptor content and hormone-responsive growth of mouse mammary tumors.

Abstract: Summary Mammary tumors were induced with estrone and progesterone in ovariectomized GR mice. Nearly all of the induced tumors were hormone dependent and remained so for the next few serial transplantations, after which they became first hormone responsive and finally hormone independent. These tumors were grafted into castrated mice that were treated with estrone and progesterone and into castrated mice that did not receive hormones. The tumors elicited in the hormone-treated castrates by hormone-dependent or hormone-responsive grafts had high estrogen receptor levels, but those obtained after grafting with hormone-independent tumors had low estrogen receptor levels. The tumors obtained in hormone-untreated castrates after grafting with hormone-responsive or hormoneindependent tumors had low estrogen receptor contents. All tumors derived from castrated hosts not given hormones were found to be hormone independent and to have low estrogen receptor contents. These results indicate that in this model system only the cells of a tumor graft that are devoid of an estrogen receptor are able to multiply in the absence of estrone and progesterone. The possible bearing of this finding on the loss of hormone responsiveness of mouse mammary tumors during successive transplant generations is discussed.

Escape from Immune Destruction by the Host through Shedding of Surface Antigens: Is This a Characteristic Shared by Malignant and Embryonic Cells?

Abstract: Summary The hypothesis is advanced that macromolecules normally found only in embryonic and fetal cells are also found in tumors because malignant cells, like the fetus, must develop mechanisms to avoid immunological destruction by the host. While anatomical factors play an important role in the “escape” of the fetus as well as the tumor, they are not by themselves adequate. In tumors, the shedding of antigens in a soluble form provides powerful protection because such antigens compete with the tumor for the effector processes of the immune response. Soluble antigens form adducts with antibodies as well as cytotoxic cells, which are then no longer capable of killing the tumor cells. Evidence is presented that the rate of spontaneous shedding of antigen may determine in part the growth pattern of the tumor in vivo. Sarcoma cells, which shed antigen rapidly, metastasize more readily than those with a slow spontaneous release of antigen. It is proposed that rapid shedding of transplantation antigens is a characteristic of embryonic cells and tumors.

Interrelationships of some chemical, physicochemical, and biological activities of several 1-(2-haloethyl)-1-nitrosoureas.

Abstract: Biological, chemical, and physicochemical data for a number of nitrosoureas have been subjected to a computer analysis to seek the relative roles of carbamoylating activity, alkylating activity, and oil solubility (octanol/water) in determining the activities of the compounds against i.p. implanted leukemia L1210. All of the compounds included in the study have some antileukemia activity, and their octanol/water distribution coefficients fall within the range designated by Hansch et al. as necessary for activity. The conclusions are as follows. Carbamoylating activity, alkylating activity, and solubility are all important in determining the degree of antileukemia activity. Perhaps a dominant role of carbamyolating activity is in determining the toxicity, as reflected in the dose (mmoles/kg) causing death of 10% of the mice when administered i.p. as a single dose and in the therapeutic indexes. Alkylating activity (which may reflect in an inverse manner the chemical half-life of the nitrosourea) is a relatively greater factor in determining the single i.p. dose (mmoles/kg) killing 99% of the leukemic L1210 cells after i.p. inoculation of 105 cells and the single i.p. dose (mmoles/kg) producing 50% 45-day survivors in a group of mice inoculated i.p. with 105 L1210 cells than in determining the dose (mmoles/kg) causing death of 10% of the mice when administered i.p. as a single dose, and it is also important in yielding desirable therapeutic indexes. The solubility (as reflected in the octanol/water distribution coefficient) is a major factor in determining the toxicity and hence the therapeutic indexes, perhaps because of the essentiality that the agents reach the critical anatomical sites for causation of toxicity. Consideration of the data leads to the suggestion that a nitrosourea having optimal therapeutic properties will have low carbamoylating activity, relatively low chemical stability, high alkylating activity, and a distribution coefficient falling at the upper end of the range for the compounds included in this study.

Childhood lymphoblastic lymphoma, a cancer of thymus-derived lymphocytes.

Abstract: Summary Populations of tumor cells obtained from children with lymphoblastic lymphoma were compared with tumor cells from children with acute lymphoblastic leukemia for thymus- or bone marrow-derived lymphocyte characteristics. Thymus derived lymphocytes were identified by their ability to bind sheep erythrocytes as rosette-forming cells. Bone marrow-derived lymphocytes were identified either by the presence of complement receptors or by the presence of immunoglobulins on their surface. Similar comparison was made between the thymus or bone marrow-derived lymphocyte properties of lymphocyte cell lines established from children with lymphoblastic lymphoma and those established from patients with other lymphoproliferative diseases. The results obtained support the notion that childhood lymphoblastic lymphoma is a cancer of thymus-derived lymphocytes and is clearly different in origin from acute lymphoblastic leukemia.

Hepatocyte proliferation and alpha 1-fetoprotein in pregnant, neonatal, and partially hepatectomized rats.

Abstract: Serum α1-fetoprotein concentrations of pregnant and newborn rats and of rats following partial hepatectomy have been measured by radioimmunoassay, and the results have been correlated to the numbers of hepatocytes synthesizing DNA. Rising concentrations follow rounds of hepatocellular proliferation indicating that the production of α1-fetoprotein is closely coupled to cellular division, i.e. , either α1-fetoprotein is synthesized transiently and released by a postmitotic cell or it is synthesized prior to mitosis and released after mitosis.

Mechanism of reaction, tissue distribution, and inhibition of arylhydroxamic acid acyltransferase.

Abstract: The enzyme of rat liver that can transform N -hydroxy- N -2-fluorenylacetamide into a reactive derivative capable of introducing fluorenylamine groups into nucleic acids has been shown to be a sulfhydryl-dependent enzyme with a molecular weight of approximately 28,000. A 30-fold purification of the enzyme from 105,000 × g supernatants of liver has been achieved by precipitation with ammonium sulfate and gel filtration on Sephadex G-100. Evidence that this mechanism of activation involves transfer of the N -acetyl group to the oxygen of the hydroxylamine came from experiments that showed that O -methylation of N -hydroxy- N -2-fluorenylacetamide prevented activation of the hydroxamic acid. Distribution studies demonstrated considerable acyltransferase activity in kidney, stomach, small intestine, and colon; lung and spleen were less active; and blood, brain, and muscle were essentially without activity. Tissue distribution studies and protein purification experiments utilizing ammonium sulfate precipitation and gel filtration techniques disclosed that the enzyme responsible for the activation of N -hydroxy- N -2-fluorenylacetamide was inseparable from the enzyme that transfers the acetyl group of N -hydroxy- N -2-fluorenylacetamide to 4-aminoazobenzene. Acyltransferase-catalyzed activation of N -hydroxy- N -2-fluorenylpropionamide demonstrated that the activation of hydroxamic acids was not restricted to acetylated derivatives. Hepatic acyltransferase-catalyzed formation of fluorenylamine-substituted nucleic acid was inhibited by both arylamines and arylacetamides. This inhibition, considered with previous reports of the inhibition of N -hydroxy- N -2-fluorenylacetamide hepatocarcinogenesis by acetanilide and p -hydroxyacetanilide, suggest that acyltransferase-catalyzed activation of arylhydroxamic acids may be involved in the formation of liver tumors.

Colon carcinogenesis in germ-free rats with 1,2-dimethylhydrazine and N-methyl-n'-nitro-N-nitrosoguanidine.

Abstract: The effect of intestinal microflora on the sensitivity of the colon to the carcinogenic effect of 1,2-dimethylhydrazine (DMH), which needs metabolic activation, and N -methyl- N ′-nitro- N -nitrosoguanidine a direct-acting carcinogen, was studied using Fischer germ-free and conventional female rats. None of the germ-free rats that received s.c. injections of DMH, 10 mg/week/kg body weight for 20 weeks and autopsied after the last injection showed colon tumors, whereas 17% of the conventional rats treated similarly developed adenocarcinomas of the colon. At 20 weeks after the last injection of DMH, 11% of germ-free rats developed adenomas, whereas 25% of the conventional rats showed colonic tumors, 67% of which were adenocarcinomas and 34% of which were adenomas. In contrast to DMH, i.r. injection of N -methyl- N ′-nitro- N -nitrosoguanidine (total dose, 48 mg in 20 weeks) nearly doubled the multiple colonic tumors in germ-free rats compared to conventional controls. It is concluded that the intestinal microflora play a modifying role in colon carcinogenesis by DMH.

The Preparation of Microsomal Fractions of Rodent Respiratory Tract and Their Characterization

Abstract: Summary A method for preparing rodent lung microsomes using normal differential centrifugation methods is described which allows the spectral characterization of these lung fractions. Further, procedures have been established to quantitate the microsomal cytochromes b 5 and P-450 in microsomes, which contain appreciable amounts of contaminating hemoglobin and methemoglobin. Physical biochemical characterizations of the rodent lung microsomal cytochromes include reduced-minus-oxidized difference spectra, low-temperature difference spectra, substrate-binding difference spectra, and enzymatic cytochrome P-450 reduction studies. In general, the lung microsomal cytochromes are similar to those found in the liver microsomes except that their specific content is 20- to 40-fold lower than in liver. The lung microsomal fraction appears to use reduced nicotinamide adenine dinucleotide more effectively as an electron donor to reduce cytochrome P-450 than do liver microsomal fractions. Administration i.p. of 3-methylcholanthrene induces the rodent lung microsomal cytochrome P-450-dependent mixed-function oxidase system by increasing the specific content of cytochrome P-450 and shifts the difference-absorption maximum of the CO derivative of reduced cytochrome P-450 from 450 nm to 448 nm. Phenobarbital treatment was without effect on the content of cytochrome P-450. Although benzphetamine demethylase and biphenyl hydroxylase activities in lung microsomes from 3-methylcholanthrene-induced animals are as high as the activities of normal liver, the lung benzo( a )pyrene hydroxylase is 400-fold less active than the comparable liver hydroxylase of control animals and the induced lung benzo( a )pyrene hydroxylase is 100-fold less active than the hydroxylase of liver microsomes from carcinogen-treated rats.

The effect of migration on the risk of nasopharyngeal cancer among Chinese.

Abstract: Summary The Chinese in California have an incidence of nasopharyngeal cancer roughly of the order of magnitude of the very high rate in Southeast Asia. Filipino migrants to California have a nasopharyngeal cancer risk intermediate to the high risk for Chinese and the low risk for Caucasians. A study of nasopharyngeal cancer mortality over the period 1958 to 1972 shows a gradient of lower risk of the disease in the second and third generation of Chinese in California. While a genetic explanation of the racial susceptibility cannot entirely be ruled out, it can be said that the Chinese are at a very high risk of the disease, partly because of unusual exposure to an environmental agent.

Relationships between the carcinogenic and mutagenic or DNA-modifying effects of nitrofuran derivatives, including 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, a food additive.

Abstract: Summary The mutagenic and DNA-modifying effects of 27 nitrofuran derivatives were studied by means of several rapid microbial assay methods. The mutagenic effects were tested with the use of Escherichia coli, B/r WP2 try- and WP2 try-, hcr-; and with Salmonella typhimurium, TA1535, TA1536, TA1537, and TA1538. The DNA-modifying effects were assayed by repair tests with E. coli, rec+, recA13, and recB21, S. typhimurium, TA1978 (uvr+) and TA1538 (uvrB). Twenty-six of the 27 nitrofuran derivatives tested had mutagenic effects on E. coli, but none of them had mutagenic effects on S. typhimurium. Twenty-five compounds had DNA-modifying effects on E. coli, and 24 compounds had effects on the DNA of S. typhimurium. Fifteen of the 27 compounds are known to be carcinogenic. Fourteen of these 15 carcinogenic derivatives had mutagenic effects on E. coli and DNA-modifying effects on E. coli and S. typhimurium. Nitrofurazone gave positive results on mutagenicity and in repair tests with E. coli, but negative results on mutagenicity and in repair tests with S. typhimurium. Three derivatives reported as noncarcinogenic also showed positive mutagenic and DNA-modifying effects. One of these, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, is widely used as a food additive in Japan. The necessity for further in vivo tests on the carcinogenicities of these three compounds is indicated. The results show that several different microbial systems must be used for detection of potential carcinogens and mutagens.

Studies on the lymphocyte 5'-nucleotidase in chronic lymphocytic leukemia, infectious mononucleosis, normal subpopulations, and phytohemagglutinin-stimulated cells.

Abstract: 5′-Nucleotidase (5′N) activity, which is present in lymphocytes isolated from the blood of normal subjects, is markedly diminished or not detectable in most patients with chronic lymphocytic leukemia (CLL). The relation to lymphocyte subpopulations and the effect of mitogenic stimulation and disease states on 5′N were investigated. The reduced activity in CLL does not stem from the increase in the percentage of B-lymphocytes found in this disorder. Stimulation of normal or 5′N-positive CLL lymphocytes with phytohemagglutinin does not lead to a decrease in activity comparable to that found in 5′N-deficient CLL cells. In contrast to what was observed with 5′N, the level of another plasma membrane marker enzyme, diphosphopyridine nucleotide diaphorase, is normal in CLL. In lymphocytes from 103 patients with a wide variety of clinical disorders, normal levels were found in all cases except during the early phase of infectious mononucleosis, when a marked decrease occurred. In certain patients with CLL the decreased enzyme level has remained constant over more than 24 months of observation; the decreased activity in infectious mononucleosis is, on the contrary, a very transient finding, with a gradual return to normal levels. The lymphoblasts isolated from the blood in 2 out of 5 patients with acute myeloblastic leukemia studied had markedly decreased 5′N activity. It appears that lymphocyte 5′ activity may be useful in the biochemical characterization of subgroups of patients with lymphoid leukemias.

A Model for the Chemotherapy of Acute Leukemia with 1-β-d-Arabinofuranosylcytosine

Abstract: The pharmacology of cytosine arabinoside (1-β-d-arabinofuranosylcytosine) is reviewed and integrated with data on the cell kinetics of normal hematopoietic and leukemic stem cells to form a model for the chemotherapy of acute leukemia. The objective of the model is to produce, by using optimal scheduling and optimal drug levels of cytosine arabinoside, a complete eradication of the leukemic stem cells during a single course of therapy by chemotherapeutic exploitation of the potential differences in the growth fraction of the leukemic and normal stem cells. The use of nutritional factors to increase the growth fraction of the leukemic stem cells is discussed in the model. Preliminary results on the effect of the model on the survival of leukemic blast cells in a patient with acute myeloblastic leukemia are reported. The use of the model to estimate the cell cycle parameters of leukemic stem cells is discussed.

Cytofluorescence Localization of Adriamycin and Daunorubicin

Abstract: Summary The characteristic orange-red fluorescence of adriamycin or daunorubicin is easily detectable in tissues of drug-treated hamsters when examined by fluorescence microscopy. Fluorescence was seen intracellularly as early as 0.5 min postinjection. Drug fluorescence was localized primarily in nuclei although some cytoplasmic drug fluorescence was detected in liver and kidney cells. No difference between adriamycin and daunorubicin was observed with this technique.

The mutagenicity and DNA-modifying effect of haloalkanes.

Abstract: Summary A series of haloalkanes, some of them widely used in industry and in the home, are shown to be mutagenic for Salmonella typhimurium and preferentially to inhibit the growth of DNA polymerase-deficient (pol A1-) Escherichia coli. It was found that the relative activities of the test substances differed when examined in these systems and that one of the agents was active in the pol A1- system only. In view of these results it is suggested that both assays be used in routine screening of environmental agents.

In vitro-in vivo studies on the susceptibility of the solid Yoshida sarcoma to drugs and hyperthermia (42 degrees).

Abstract: Summary Over 2000 allogeneic solid Yoshida tumors of rats were studied to examine the predictive value of in vitro testing for tumor sensitivity to drugs and hyperthermia. In vitro , the alkylating agent methylenedimethanesulfonate (MDMS) caused a concentration-dependent inhibition of respiration, anaerobic glycolysis, and radioactive thymidine, uridine, and leucine uptake into cultures of the tumor over 24 hr. Hyperthermia (42°) had a progressive inhibitory effect on these parameters over 1 to 4 hr. MDMS potentiated the effect of 42° temperature on the tumor in vitro . Tumor regression correlated with as little as 25 to 30% inhibition of respiration by the drug in vitro; inhibition of respiration also provided a reliable and rapid index of in vivo response to hyperthermia. In vivo , exposure to 42° for 1 hr led to disappearance of 1.5-ml foot tumors within 12 to 14 days, and MDMS (2.5 mg/kg) caused significantly more rapid tumor regression within 8 to 10 days. MDMS potentiated the effect of heat on tumor regression. Biochemical and histological data showed that in the animal the two therapies had different modes of action not predicted by the in vitro system. Following MDMS, there was tumor cell pyknosis, fibroblast infiltration, and a rapid decrease in tumor volume, accompanied by progressive decrease in respiration, glycolysis, and isotope uptake in the regressing tumor. Hyperthermia resulted in less rapid cell death, slower removal of dying cells, and more gradual replacement of tumor architecture by connective tissue. This was associated with a more protracted decrease in isotope uptake and an inhibition of respiration; glycolysis was depressed only temporarily. Other cytotoxic drugs (except alkylating agents) were ineffective, both in vitro and in vivo . The results indicate that in vitro testing can be of predictive value for assessing the response of a tumor in the host to drug or heat therapy.