The resolution of mixtures of viable mammalian cells into homogeneous fractions by zonal centrifugation.

Large-scale separation of mixtures of mammalian cells was obtained with the A-1X zonal centrifuge rotor and density gradients consisting of Ficoll dissolved in modified Eagle's MEM suspension-culture medium. The cells remained viable as tested by plating efficiency or by motility observed with time-lapse photography. Rabbit thymocyte and HeLa cell mixtures were separated with 99 and 89 per cent purity, respectively. Mixtures of thymocytes and suspension-cultured, human acute leukemia cells (Roswell Park strain LKID) were separated with 93 and 91% purity, respectively. HeLa cells were isolated 92% pure from a mixture with horse leukocytes. A book of charts giving the sedimentation position and velocity versus time of cells in the A rotor under standard conditions of gradient composition, angular velocity, and temperature was prepared with the use of a computer program based on the differential sedimentation equation. The charts are used to estimate the centrifugation time necessary for maximum separation of cells. The success achieved in separating mixtures of cells points to the future possibility of large-scale fractionation of solid tissues, especially tumor tissues, into preparations cf viable cells of a single type.

Isolation of plasma membrane fragments from HeLa cells.

A method for isolating plasma membrane fragments from HeLa cells is described. The procedure starts with the preparation of cell membrane "ghosts," obtained by gentle rupture of hypotonically swollen cells, evacuation of most of the cell contents by repeated washing, and isolation of the ghosts on a discontinuous sucrose density gradient. The ghosts are then treated by minimal sonication (5 sec) at pH 8.6, which causes the ghost membranes to pinch off into small vesicles but leaves any remaining larger intracellular particulates intact and separable by differential centrifugation. The ghost membrane vesicles are then subjected to isopycnic centrifugation on a 20-50% w/w continuous sucrose gradient in tris-magnesium buffer, pH 8.6. A band of morphologically homogeneous smooth vesicles, derived principally from plasma membrane, is recovered at 30-33% (peak density = 1.137). The plasma membrane fraction contained a Na-K-activated ATPase activity of 1.5 micromole Pi/hr per mg, 3% RNA, and 13.8% of the NADH-cytochrome c reductase activity of a heavier fraction from the same gradient which contained mitochondria and rough endoplasmic vesicles. The plasma membranes of viable HeLa cells were marked with (125)I-labeled horse antibody and followed through the isolation procedure. The specific antibody binding of the plasma membrane vesicle fraction was increased 49-fold over that of the original whole cells.

Malignant hemangioendotheliomas produced by subcutaneous inoculation of Balb/3T3 cells attached to glass beads.

The Balb/3T3 mouse embryo cell line has been frequently used in cancer research as representative of nontumorigenic cells with the characteristic in vitro properties of postconfluence inhibition of cell division, low saturation density, and anchorage dependence. On the reasoning that anchorage dependence might also apply in vivo, each of nine mice were subcutaneously inoculated with an average of 15,400 Balb/3T3 cells attached to two glass beads 3 millimeters in diameter. After 8 weeks, all the mice had developed large bloody tumors that microscopically proved to be hemangioendotheliomas. Ther inoculation of Balb/3T3 cells alone or beads alone produced no tumors. Transplants of each tumor into normal mice grew to kill the animal within 6 weeks. Tumor cells from collagenase-disaggregated tumor tissue had a plating efficiency of 21.2 percent compared to that of normal adult subcutaneous fibroblasts of less than 0.1 percent. The tumor cells in vitro closely resembled Balb/3T3 cells in appearance and were tumorigenic at a dose of 10-4 cells. A second, repeat experiment produced the same type of tumors grossly and microscopically in 17 of 25 mice between 99 and 211 days after inoculation of the Balb/3T3 cells attached to glass beads. These findings require a reassessment of the postulate that low saturation density, postconfluence of cell division, and anchorage dependence are characteristic in vitro properties only of nonneoplastic cells.

Centrifugation of mammalian cells on gradients: a new rotor.

A short-arm rotor increases separation of viable mammalian cells, from mixtures, by low-speed centrifugation; continuous Ficoll density gradients in tissue-culture media are used. We describe the theory and experimental demonstration of the superior separation achieved with this new rotor.

"Sontaneous" neoplastic transformation in vitro: a form of foreign body (smooth surface) tumorigenesis.

Explants of subcutaneous connective tissue from adult BALB/c mice into plastic petri dishes were serially subcultured and tested for tumorigenicity in two ways: by the subcutaneous implantation of cells attached to plastic plates (1 by 5 by 10 millimeters), and by the subcutaneous injection of cells suspended in saline. Cells grown in vitro for 18 or more days before being implanted attached to a plastic plate (2.4 x 10(4) to 3.4 x 10(5) cells per plate) formed tumors after 24 to 79 weeks. The latent period before tumor appearance correlated inversely with the time spent by the cells in tissue culture. Cells inoculated in saline suspension (10 to 100 times the above number per plate) did not form tumors until after 84 days in vitro; plates alone did not induce tumor formation within more than 1 1/2 years of implantation. The tumors arising from the plate-attached cells were transplantable without plates and histologically appeared to be undifferentiated sarcomas. It is well established that smooth-surfaced foreign bodies, regardless of their chemical composition, will produce sarcomas when transplanted subcutaneously in rodents. We interpret our data, particularly the decrease in tumor latent period with time spent in tissue culture, as indicating that a smooth surface was acting as a carcinogen first in vitro (the surface of the tissue culture dish) and then in vivo (the surface of the plastic plate).

Local adoptive transfer of the antitumor cellular immune response in syngeneic and allogeneic mice studied with a rapid radioisotopic footpad assay.

The immunologic nature of the cellular immune response against tumor cells inoculated in the footpad of mice was studied with a rapid, quantitative, and specific assay. The results indicate: a) The antitumor cellular immune response could be transferred adoptively in syngeneic and allogeneic mice with specific immune thymus (T) lymphocytes isolated on nylon columns; b) T-independent cells of host origin were necessary for the manifestation of the antitumor footpad reaction; and c) there was a close correlation between immune responses detected by the footpad assay and those detected by transplantation techniques. The footpad reaction consisted of several nonspecific and specific components. Nonspecific factors disturbing the specific footpad reaction in syngeneic and allogeneic recipients were discussed.

Inhibition of the cellular immune response to simian virus 40 tumor cells in tumor-bearing and tumor-immune mice by concanavalin A.

The effects of in vivo-administered concanavalin A (Con A) on the kinetics of the primary and secondary cellular immune responses to simian virus 40-transformed tumor cells were investigated in BALB/c mice. Either a single initial dose of 400 mug Con A or daily doses of 50 mug depressed the cell-mediated immune response to tumor cells during the progressive growth of tumors, as determined by a radioisotopic foot-pad assay. The immune depression correlated with an increase in ultimate tumor weight. Similarly, Con A suppressed the antitumor cellular immune response in tumor-immune animals. Immune reactivity returned within 6 days after a single injection of 400 mug Con. Continuous administration 50 mug Con A resulted in a gradual decline in antitumor cellular immune responsiveness, which reached a plateau by the 5th day. Splenic lymphocytes from Con A-treated, immune mice failed to elicit a local adoptive transfer reaction; their immune responsiveness tended to return after incubation with alpha-methyl-D-pyranosyl sugars.

PRomotion of smooth surface tumorigenicity by phorbol myristate acetate in Balb/3T3 cells and Balb/3T3 T proadipocytes.

In vitro exposure of Balb/3T3 cells to phorbol and phorbol myristate acetate significantly increased their tumorigenic potential when implanted sc on smooth surface plates into syngeneic mice. This finding supports the hypothesis that many so-called normal cell lines may actually represent initiated cells that can be induced to become tumorigenic following exposure to promoting agents. Since the tumorigenic potential of many tissues in vivo is inversely proportional to the state of differentiation of the stem cell populations undergoing transformation, we assayed the relative tumorigenicity of Balb/3T3 T proadipocytes, which can differentiate in culture, and Balb/3T3 cells, which cannot differentiate in culture. The effects of tumor-promoting agents on these cells were also tested. Plate-implanted Balb/3T3 T proadipocytes were markedly less tumorigenic than Balb/3T3 cells, and Balb/3T3 T proadipocytes were not sensitive to the promotional effects of phorbol myristate acetate.

Local adoptive transfer of antitumor cellular immunity to xenogeneic animals studied with a rapid radioisotopic footpad assay.

The antitumor cellular immune response to Gross virus-induced rat tumor cells in F344 rats, as measured by a sensitive radioisotopic footpad assay, was adoptively transferred to syngeneic rats and to xenogeneic irradiated BALB/c mice. Xenogeneic transfer was accomplished by the injection of a mixture of rat tumor cells and syngeneic spleen cells, peritoneal exudate cells, or blood lymphocytes from specific immune rats into the footpads of mice. Peritoneal exudate cells produced the strongest footpad reaction in xenogeneic recipients. Use of the xenogeneic adoptive transfer system in a bioassay for human antitumor immunity appeared feasible.

Anticancer drugs: effect on the cloning of Raji lymphoma cells in soft agar.

The effect of 11 anticancer drugs on the ability of Raji lymphoma cells to form colonies in soft agar was determined with the use of both a 1-hour and a continuous drug exposure. Three distinct patterns of drug sensitivities were observed: a) Dactinomycin, adriamycin, bleomycin, mitomycin C, vincristine, and cis-platinum II all produced a dose-dependent reduction in colony formation following a 1-hour exposure, which was further augmented by a continuous exposure to the drugs; b) the antimetabolites (methotrexate, beta-cytosine arabinoside, and 5-fluorouracil) and pentamethylmelamine had no suppressive effects on colony formation with a 1-hour exposure, but they produced marked cytotoxicity with continuous drug exposure; and c) L-phenylalanine mustard had the same degree of colony suppression with both a 1-hour and a continuous drug exposure. Preincubation of Raji cells with an enzyme mixture (DNase + pronase + collagenase) did not alter the degree of colony suppression observed with the anticancer drugs. These results indicate that continuous drug exposure should be compared to a 1-hour drug incubation to determine in vitro drug sensitivities of fresh human tumors in the soft agar clonogenic assay, because the 1-hour drug exposure may not identify certain drugs that are potentially clinically active.

Changes in the mitogen response of lymphoid cells with progressive tumor growth.

The response of spleen and lymph node cells from tumor-bearing animals to a variety of mitogens was measured. It was found that progressive tumor growth diminished the reactivity of spleen cells to the mitogen phytohemagglutinin. Lymph node cells from the same animal were not consistently so affected. The reactivity of spleen cells from tumor-bearing donors to concanavalin A was somewhat depressed, while stimulation by bone marrow-derived lymphocyte mitogens such as endotoxin and pokeweed mitogen was in some cases enhanced. Theta antigen expression in tumor bearer spleens was found to decline steadily after 7 days of tumor growth. Cytological analysis revealed that the normal structure of the spleens of tumor animals was infiltrated by myeloid elements. The changes described occurred regardless of whether the tumors were chemically or virally induced. Excision of tumors after long periods of growth resulted in prompt return of splenic phytohemagglutinin sensitivity. The data suggests that loss or incapacitation of parts of the normal thymus-derived lymphocyte components may occur in the spleens of animals with progressive neoplastic growth.

Specific depression of the antitumor cellular immune response with autologous tumor homogenate.

A momogenate of an SV40-transformed firbosarcoma of BALB/c mice (E4 tumor) injected i.p. into E4, tumor-immune syngeneic mice specifically depressed their cell-mediated immune responses to autologous tumor cells, as measured by a radioisotopic foot pad assay. The fraction of the tumor homogenate that brought about this depression was present in the high-speed supernatant and pellet of a 3 M KCl extract of the tumor. The specificity of the depression was shown in three ways: (a) the serum of E4 tumor-immune mice, but not of normal mice, given injections of E4 tumor homogenate 24 hr previously, suppressed antitumor immunity in vitro, as measured by the release of 51Cr from labeled E4 tumor cells incubated with spleen cells from tumor-immune animals; (b) the i.p. inoculation of E4 tumor homogenate did not alter the cellular immune response of tuberculin-sensitized mice to tuberculin; and (c) the i.p. injection of a homogenate of antigenically unrelated tumor did not depress the cellular immune response of E4 tumor-immune mice to E4 tumor cells.

Common tumor rejection antigens in methylcholanthrene-induced squamous cell carcinomas of mice detected by tumor protection and a radioisotopic footpad assay.

Seven transplantable lines of squamous cell carcinoma of the skin initiated in BALB/c mice by skin painting with methycholanthrene were systematically tested for cross-reactivity of their tumor rejection antigens in a 7 X 7 matrix. As determined by decreased tumor frequency after tumor cell challenge, each line was immunogenic against and/or immunosensitive to at least one and usually more than one of the other lines. A radioisotopic footpad assay for delayed hypersensitivity against viable tumor cells confirmed the cross-reactivity shown by tumor rejection. More than two antigens appeared to be present in the lines. Tests for C-type viruses were positive in all tumors; those for polyoma virus were negative. Whether the uniform presence of C-type viruses can account for the number and variety of antigens found, or whether the tumor rejection antigens are independent of virus expression, remains an open question. The finding of cross-reacting tumor rejection antigens in methylcholanthrene-induced squamous cell carcinomas encourages prospects for the development of more broadly applicable immunodiagnostic and immunotherapeutic reagents.

Identification of candidate cancer chemopreventive agents and their evaluation in animal models and human clinical trials: a review.

A search of the literature using National Library of Medicine databases and individual cancer journal articles yielded over 500 compounds with published chemopreventive activity in animals. From these, an initial 16 agents or agent combinations have been evaluated in the following animal tumor models: mouse skin papillomas/carcinomas induced by 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate; rat breast adenocarcinoma induced by N-methyl-N-nitrosourea or 7,12-dimethylbenz(a)anthracene; hamster lung carcinoma induced by N-methyl-N-nitrosourea or diethylnitrosamine; mouse bladder papillary carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine; and rat and mouse colon cancer induced by azoxymethane/methylazoxymethanol acetate. Some of the most interesting positive results observed include 4-hydroxyphenyl retinamide plus tamoxifen in breast cancer, piroxicam in colon cancer, dimethylfluoroornithine in breast and bladder cancer, oltipraz in lung cancer, dehydroepiandrosterone in colon cancer, and molybdate in bladder cancer. Eighteen human intervention trials in progress are described that involve the following agents: beta-carotene (eight trials). Retinol/retinoic acid (seven trials), vitamins C and E (three trials), 4-hydroxyphenyl retinamide (one trial), piroxicam (one trial), and calcium (one trial). By organ site these studies involve cancer of the lung (six studies), skin (five studies), colon (four studies), breast (one study), and uterine cervix (two studies).

Natural history of intraepithelial neoplasia in humans with implications for cancer chemoprevention strategy.

Intraepithelial neoplasia is of critical importance to the cancer chemoprevention field because it is a target condition for which drugs must be sought that will prevent its development or stop its progression. The term "dysplasia" refers to the morphological alterations that characterize intraepithelial neoplasia and according to many authors consists of seven basic morphological changes that occur in the majority of human epithelia, as well as in the epithelium of mouse skin papillomas induced by 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanoylphorbol-13-acetate: increased nuclear size; altered nuclear shape; increased nuclear stain uptake; nuclear pleomorphism (increased variation in nuclear size, shape, and stain uptake); increased mitoses; abnormal mitoses; and disordered or absent maturation. Clonal evolution appears to begin early in the neoplastic process during intraepithelial neoplasia. Aneuploidy has been found during intraepithelial neoplasia in many human epithelia, and, in association with other forms of genetic instability, may provide the increase in genetically variant cells required for clonal evolution to occur. It is postulated that two major factors affecting the rate of progression of intraepithelial neoplasia are the cellular mutation rate, which is enhanced by environmental carcinogens, and the cellular proliferation rate, which is enhanced by agents that include sex hormones, inducers of chronic inflammation, and irritant chemicals which stimulate reactive hyperproliferation. A preferred chemoprevention strategy should consist of the development of drugs and drug combinations which will block mutagenic carcinogens or prevent epithelial hyperproliferation or its causes. Two examples of the induction of regression of intraepithelial neoplasia by chemopreventive drugs are the regression of oral leukoplakia produced by beta-carotene and the regression of colorectal polyps in patients with familial polyposis produced by sulindac. It is evident that there is a strong need for more research on the induction of regression of intraepithelial neoplasia with chemopreventive agents. There is also a critical need to identify and develop biomarkers that correlate with the appearance and regression of intraepithelial neoplasia.

Vasoformative sarcomas arising from BALB/3T3 cells attached to solid substrates.

The BALB/3T3 mouse embryo cell line, noted for its marked postconfluence inhibition of proliferation, anchorage dependence, and high serum requirement, and frequently studied as a prototype nontumorigenic "fibroblast" line that is compared with tumorigenic sublines transformed with various agents, produced tumors within 2 to 3 months when an average of 3 X 10(4) cells were implanted s.c. attached to 1- X 5- X 10-mm polycarbonate platelets. Plastic platelets alone produced no tumors after 1 year of observation. The tumors, as well as others arising from implants of BALB/3T3 cells attached to 3-mm glass beads, were given the histological diagnosis of "vasoformative saroma" because the tumor cells frequently formed vascular channels. The vasoformative pattern and the results of specific staining for reticulin and collagen support the likelihood that BALB/3T3 cells originated from endothelial cells rather than from fibroblasts. That the tumors were derived from BALB/3T3 cells and not host cells was proved when tumors arising in BALB/c X C57BL/6 F1 hybrids were shown to be transplantable to BALB/c but not to C57BL/6 mice. The cultured tumor cells showed loss of both postconfluence inhibition of proliferation and anchorage dependence. Evidence of the induction of endogenous oncornaviruses was obtained in only one of four tumors tested. These tumors also exhibited tumor-unique transplantation rejection antigens. We conclude that BALB/3T3 cells are preneoplastic and give rise to different spontaneously transformed clones bearing unique tumor rejection antigens when implanted in vivo attached to a solid substrate.

Accelerated regeneration of trypsin-treated surface antigens of simian virus 40-transformed BALB/3T3 cells induced by X-irradiation.

The antigens of SV40-transformed BALB/3T3 cells measured by a radioisotopic footpad assay after removal by trypsin treatment regenerated in vitro in 3 to 6 hr. After X-irradiation with 3000 R, however, the antigens were regenerated to normal levels within 1 h. X-ray doses of between 1000 and 5000 R accelerated the regeneration of cell surface antigens, while X-irradiation with the larger dose of 8000 R did not. X-irradiation of nontrypsinized tumor cells was without effect. Possible mechanisms of this phenomenon are discussed.

Augmented immunogenicity of tumor cell membranes produced by infection with influenza virus as compared to Moloney sarcoma virus.

The tumor-associated transplantation antigens (TATA) of crude membrane extracts from SV40-transformed BALB/3T3 tumor cells lytically infected with influenza virus were markedly more immunogenic than were extracts from uninfected cells measured either by the ability to induce heightened resistance to tumorgraft challenge or by heightened lymphocyte-mediated cytotoxicity against tumor cells in vitro. When intact tumor cells (as opposed to membrane extracts) were productively infected with Moloney sarcoma virus, they were made so immunogenic that they would only grow in X-irradiated syngeneic animals. Yet crude membrane extracts from the Moloney sarcoma virus-infected tumor cells showed no increase in TATA activity analogous to that seen after infection with influenza virus. Thus, influenza virus augmentation of tumor membrane TATA may operate by a different mechanism than does the oncornavirus augmentation of intact tumor cell TATA reported by others. It appears that Moloney sarcoma virus and possibly other oncornaviruses cannot be used to augment the TATA activity of tumor cell membranes in the same way that other surface-budding viruses can.

Adoptive immunotherapy of a Gross virus producing lymphoma and a methylcholanthrene-induced fibrosarcoma in tolerant rats.

Immunological tolerance to Gross virus-specific transplantation antigens in rats given neonatae transfer of donor lymphoid cells beneath the kidney capsule of syngeneic recipient rats. Immune or normal donor cells invariably developed a cell-mediated immune reaction in kidneys of GV-tolerant recipients, presumably against GV antigens present on the surface of recipient lymphoid cells in the kidney. Spleen and lymph node cells from tolerant rats failed to develop a reaction in tolerant recipients, but developed a strong reaction to histoincompatible antigens in the kidneys of semisyngeneic tolerant rats. The immunologically tolerant state in the rats could be broken by adoptive transfer of spleen and lymph node cells from syngeneic rats immunized with GV-induced lymphoma cells. Immunotherapy of a GV-induced and also a GV-infected methylcholanthrene-induced fibrosarcoma growing in tolerant rats was successful when immune spleen and lymph node cells were administered i.p. 3 days after s.c. inoculation of 2 X 10(7) tumor cells in the case of the lymphoma, and 1 day after inoculation of 5 X 10(6) tumor cells in the case of the fibrosarcoma.

Karyotypes of vasoformative sarcomas arising from BALB/3T3 cells attached to polycarbonate plates.

Four vasoformative sarcoma in vivo tumor lines arising from the s.c. implantation of 3 X 10(4) BALB/3T3 cells attached to a 1- X 5- X 10-mm polycarbonate platelet were shown to be quite similar in chromosome constitution to the parental 3T3 line. All tumors contained the same marker chromosomes that were present in the parent BALB/3T3 cells. The findings conclusively showed that the tumors had derived from BALB/3T3 cells and not from host cells or from the in vivo fusion of the inoculated 3T3 cells with host cells. Each tumor line had a unique karyotype that was markedly more homogeneous than that of the parental BALB/3T3 cells, strongly suggesting that each tumor represented a separate clone. An M1 marker chromosome was consistently present in each of the four tumor lines but was present in only about 50% of the parent BALB/3T3 cells; it therefore appeared to be a distinct and stable feature of the tumors.