Plasma membrane-associated phosphatase activities hydrolyzing [32P]phosphotyrosyl histones and [32P]phosphatidylinositol phosphate.

We describe a procedure of preparing [32P]phosphotyrosyl histones with minimal contamination by 32P-labeled lipids; the latter was usually found to be mixed with the phosphoproteins when the cell membrane-enriched fraction of A-431 cells was used as a source of tyrosine kinase. The phosphatase activities previously found to be associated with the plasma membranes of a human astrocytoma were resolved using purified [32P]phosphotyrosyl histones and [32P]phosphatidylinositol phosphate. In comparison with the phosphotyrosyl protein phosphatase, the phosphatidylinositol phosphate phosphatase activity is more active over a broad range of pH values, and its activity is inhibited by fluoride, zinc chloride, and lower concentrations of vanadate.

Spurious DNA blot hybridization resulting from bacterial contamination of primary tissue preparations.

We have observed, by Southern blot hybridization, numerous episomes in DNA prepared from tumors grown as athymic mouse xenografts. These extrachromosomal DNAs were present in multiple copies and existed as relaxed and supercoiled conformational isomers. The episomes were readily detected with pBR322 plasmid probes, but not with purified plasmid inserts. Subsequently, four species of bacteria were isolated from tumor xenografts, suggesting that the pBR322 related episomes which we observed were bacterial DNAs, copurified during the isolation of xenograft DNA. This finding illustrates a potential problem which may be encountered in blot hybridizations utilizing nucleic acids from primary tissue preparations.

Activity of cancer chemotherapeutic agents against human colorectal carcinomas grown as primary tissue culture.

Improved procedures are described for the seeding of primary cultures from human colon adenocarcinoma and for the use of these cultures in the evaluation of drug effects. Two of the specimens studied were xenografts maintained in athymic (nude) mice, while the other six were biopsies obtained directly from patients. Tumor cells obtained directly from the patients proliferated in defined hormone-supplemented medium to the exclusion of other cells. In drug-response studies with cultures from a colon tumor biopsy all four drugs studied (4'-deoxydoxorubicin, 4'-O-methyldoxorubicin, 5-fluorouracil, and 1,3-bis-[chloroethyl]-1-nitrosourea) inhibited growth of the cells within 3-6 days after drug treatment. On an equitoxic dose basis (LD10 in mice), 4'-deoxydoxorubicin appeared to be the most active drug. This drug also showed dose-dependent activity against one of the xenografted tumors in vitro. In dose-response studies with cultures from another patient's colon tumor, doxorubicin and 5-fluorouracil showed significant activity against the tumor 10 days after the drug treatment with concentrations at 1X and 10X average peak plasma levels.

Kinetics of protein synthesis inactivation in human T-lymphocytes by selective monoclonal antibody-ricin conjugates.

Immunotoxins synthesized with the pan-T-cell monoclonal antibody T101 and ricin, acetylricin, or ricin A-chain have been compared. Native ricin was acetylated with N-acetylimidazole to block the galactose-binding site of the toxin B (binding)-chain. In the presence of lactose, both whole-ricin-containing immunotoxins were selectively cytotoxic but the ricin A-chain conjugate was less effective in blocking cellular protein synthesis. Immunotoxin-treated cells cultured in fresh growth medium exhibited no growth, declining viabilities, and no protein synthesis activity. Lymphocytes treated with T101:ricin or ricin did not form clusters or colonies when plated in 0.3% Bacto-agar. Ammonium chloride markedly enhanced the efficacy of T101:ricin and T101:ricin A-chain. Our results suggest that: (a) all immunotoxins were selectively cytotoxic; (b) in the presence of ammonium chloride the effectiveness of the T101:ricin A-chain conjugate approached that of T101:ricin; and (c) the toxin B-chain may facilitate conjugate internalization and/or processing.

Demonstration of separate phosphotyrosyl- and phosphoseryl- histone phosphatase activities in the plasma membranes of a human astrocytoma.

A plasma membrane preparation from a human astrocytoma contained p-nitrophenyl phosphate (pNPP), phosphotyrosyl histone, and phosphoseryl histone hydrolysis activities. The pNPPase and phosphotyrosyl histone phosphatase activities were inhibited by vanadate, whereas the phosphoseryl histone phosphatase activity was not; the latter activity was inhibited by pyrophosphate and nucleoside di- and triphosphates. When the membranes were solubilized by Triton X-100 and the solubilized proteins were subjected to column chromatography on DEAE-Sephadex, Sepharose 6B-C1, and wheat germ agglutinin-Sepharose 4B columns, the pNPPase activity from the phosphoseryl histone phosphatase activity. The results from column chromatography also indicated that there may be multiple phosphotyrosyl and phosphoseryl protein phosphatases in the plasma membranes.

A leukemia virus-related protein in the murine pancreas.

A protein that has been detected in the granules of islet cells in the murine pancreas is similar but not identical to the endogenous murine leukemia virus envelope protein gp70. The pancreatic protein was detected by several immunological methods using both polyclonal and monoclonal anti-murine gp70. On purification by affinity chromatography, it was shown to be different from murine gp70 in its subcellular location and its molecular size and the size of its precursor and by the effect of various reagents on its immunological activity as determined by the ELISA assay.

Amplification of the murine leukemia virus Mr 70,000 glycoprotein gene product by human xenografts in athymic mice.

Passage of human tumors in athymic mice is accompanied by an increase in serum levels of the Mr 70,000 murine leukemia virus envelope protein, gp70. Elevated levels of gp70 can be detected in tissues of the hematopoietic systems of mice bearing human xenografts, but there is no evidence of synthesis of gp70 in these tissues. By far, the highest concentration of gp70 is in the human xenografts themselves. When assayed for gp70, 8 human xenografts and 12 cell lines established from human xenografts were all positive. In the plasma membrane of the human astrocytoma xenograft, T24, the gp70 was found to be approximately 10% of the total membrane protein. In contrast, the concentration of the Mr 30,000 viral core protein, p30, was 17-fold less. Only trace amounts of complete infectious virus could be detected. A human prostate carcinoma line that had not been grown in the athymic mice was found to have no gp70, but was shown to be able to synthesize gp70 after a single passage in the athymic mice.

Correlation of drug sensitivity on human colon adenocarcinoma cells grown in soft agar and in athymic mice.

A well-differentiated colorectal tumor T 219 which grows as a xenograft in athymic mice (human-tumor-nude-mouse system) and forms colonies in culture (soft agar colony-formation assay) has been used to test the correlation between the above two methods of exposure of human tumor cells to antineoplastic agents. In in vitro studies, two protocols were used: 1 h drug exposure and continuous drug exposure. In the 1 h drug exposure experiments six drugs, doxorubicin (DX), 4'-deoxydoxorubicin (deoDX), 4'epidoxorubicin(epiDX), 4'-O-methyldoxorubicin (O-DX), N-trifluoroacetyldoxorubicin-14-valerate (AD-32) and 5-fluorouracil (FUra) were studied, while in continuous drug exposure experiments four of the above drugs (DX, deoDX, epiDX, O-DX) were studied. The survival of the tumor clonogenic cells (HC219) was determined by counting the number of colonies formed during 13-14 days of incubation and dose-response curves were obtained. In in vivo studies, the mice were treated with all of the drugs used in in vitro 1 h drug exposure experiments (DX, deoDX, epiDX, O-DX, AD-32 and FUra). To quantitate the chemotherapeutic effectiveness of the drugs, T/C% (relative tumor volume of treated group as percentage of the control group) values were calculated each time the tumors were measured. The experimental data suggest that in vitro 1 h drug exposure results are in good agreement with the in vivo results, while the continuous drug exposure results do not agree with the in vivo data. The most active drug in in vivo studies, deoDX, was found to be the most active drug in the in vitro 1 h drug exposure experiments as well. However, in continuous drug exposure experiments, O-DX, not deoDX, was found to be the most active drug. Activities of the other drugs tested also differed from their respective activities in in vivo studies. Although the relative effectiveness of various drugs can be compared by determining molar concentrations of the drugs producing 50% inhibition of colonies (ID50) the expression, PEI = LD10/ID50 X 1000, which takes into consideration toxicity of the drugs, is probably a better indicator of the in vitro drug activity. The results suggest that soft agar colony-formation assay (with established cell lines from the same tumor) may be used for the prediction of in vivo activity of potential antineoplastic agents against human tumor xenografts in nude mice.

Monoclonal antibody and an antibody-toxin conjugate to a cell surface proteoglycan of melanoma cells suppress in vivo tumor growth.

A monoclonal antibody directed against a cell surface chondroitin sulfate proteoglycan of human melanoma cells, 9.2.27, and its diphtheria toxin A chain (DTA) conjugate were investigated for their effects on in vitro protein synthesis and in vivo tumor growth of human melanoma cells. The 9.2.27 IgG and its DTA conjugate display similar serological activities against melanoma target cells but only the conjugate can induce consistent in vitro inhibition of protein synthesis and toxicity in M21 melanoma cells. However, both 9.2.27 IgG and its DTA conjugate effect significant suppression of M21 tumor growth in vivo in an immunotherapy model of a rapidly growing tumor in athymic nu/nu mice, suggesting that other host mechanisms may mediate monoclonal antibody-induced tumor suppression.

An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.

In vitro responses of nude mouse-xenografted human colon carcinomas exposed to doxorubicin derivatives in tissue culture and in the mouse.

In vitro responses of 4 xenografted human colon tumors (T183, T219, T245, and T348) to various doses of 4'-deoxydoxorubicin have been investigated. The individual tumors showed marked differences in drug responsiveness, ranging from high sensitivity at low doses (T219; 125 ng/ml) to very low sensitivity at high doses (T245; 4000 ng/ml). The sensitivity ranking deduced from these in vitro experiments correlates well with the ranking deduced earlie (Guiliani et al., Int. J. Cancer, 27: 5-13, 1981) from in vivo drug treatments of transplants of these tumors in the nude mouse. The effect of in vitro drug treatment (4'-deoxydoxorubicin; 250 ng/ml; 1-hr incubation) on the in vivo growth of one of the tumors, T219, in nude mice was investigated. Growth of the tumor in nude mice was markedly delayed by pretreatments in vitro with 4'-deoxydoxorubicin. Furthermore, in vitro responsiveness of the T219 tumor was investigated following in vivo and in vitro treatment of the tumor with 4'-deoxydoxorubicin. Both of the pretreatments produced very similar decreases in drug responsiveness to all of the doxorubicin derivatives tested (4'-deoxydoxorubicin, 4'-O-methyldoxorubicin, 4'-epidoxorubicin, 4-demethoxydoxorubicin, and N-trifluoroacetyldoxorubicin-14-valeriate).

Differentiation and activation of nu/nu splenic T cell precursors by mature peripheral T cells in the absence of thymus.

Nu/nu splenic T cell precursors lacking significant Thy-1 surface antigen are driven in vitro to proliferate and express Thy-1 during incubation with activated, nondividing peripheral T cells in the absence of thymus or thymic extracts. The precursors are present in nu/+ spleen as well, and are phenotypically similar to thymocyte precursors assayed in vivo. The nondividing inducer cells required are Lyt-2-, Thy-1+ T cells present in nu/+ but not nu/nu spleen and show no MHC restriction in the induction process. Using this in vitro assay, we preliminarily identified a monoclonal rat anti-mouse brain antibody that lyses nu/nu responding T cell presursors in the presence of complement. The ability of mature peripheral T cells to induce the differentiation and activation of T cell precursors in the complete absence of thymus appears to explain the grossly different effects of neonatal and adult thymectomy or thymic involution on immunocompetence. In addition, we showed that the limiting cell in three-cell mitogenic response of normal spleen cells to Con A is likely the macrophage by similar analysis of nondividing inducer cell requirements. This finding allows the assignment of a unique order to this three-cell response.

Induction of sarcomas in athymic mice.

During the course of serial passage of 50 human xenografts in the athymic mouse over a period of 5 years we have observed two cases of induction of sarcomas in the murine stromal tissue associated with the human xenografts. Both times the growth of the murine sarcomas overtook that of the human xenograft. This change was monitored by analysis of the lactate dehydrogenase isozyme profile and histology of each passage of the human xenografts in the athymic mice. The two murine sarcomas were subsequently established in tissue culture. The sarcoma cell lines were found to be malignant by morphological and growth characteristics and were tumorigenic. They contained large amounts of murine leukemia virus when assayed for reverse transcriptase activity by infection of mouse SC-1 cells and BALB/c and NIH Swiss fibroblasts with filtered supernates, and some type C virus particles were observed by electron microscopy in tumor tissues. However, we were unable to demonstrate the presence of murine sarcoma virus by in vitro transformation of fibroblasts or sarcoma formation in vivo will cell free filtrates. Preliminary biochemical data indicate that the sarcomas are extremely high in plasma membrane ATP phosphohydrolase.

The activity of the acidic phosphoproteins from the 80 S rat liver ribosome.

The selective removal of acidic phosphoproteins from the 80 S rat liver ribosome was accomplished by successive alcohol extractions at low salt concentration. The resulting core ribosomes lost over 90% of their translation activity and were unable to support the elongation factor 2 GTPase reaction. Both activities were partially restored when the dialyzed extracts were added back to the core ribosome. The binding of labeled adenosine diphosphoribosyl-elongation factor 2 to ribosomes was also affected by extraction and could be reconstituted, although not to the same extent as the GTPase activity associated with elongation factor 2 in the presence of the ribosome. The alcohol extracts of the 80 S ribosome contained mostly phosphoproteins P1 and P2 which could be dephosphorylated and rephosphorylated in solution by alkaline phosphatase and protein kinase, respectively. Dephosphorylation of the P1/P2 mixture in the extracts caused a decrease in the ability of these proteins to reactivate the polyphenylalanine synthesis activity of the core ribosome. However, treatment of the dephosphorylated proteins with the catalytic subunit of 3':5'-cAMP-dependent protein kinase in the presence of ATP reactivated the proteins when compared to the activity of the native extracts. Rabbit antisera raised against the alcohol-extracted proteins were capable of impairing both the polyphenylalanine synthesis reaction and the elongation factor 2-dependent GTPase reaction in the intact ribosomes.

Induction of lymphoma in antigenically stimulated athymic mice.

Athymic mice infected with pinworms or carrying human tumor xenografts frequently develop a lymphoproliferative disorder which eventually leads to lymphoma. By immunofluorescent analysis of involved tissues, the lymphomas appear to be mixtures of null cells, B-cells, and T-cells. When each lymphoma is established in tissue culture, a predominant cell type grows out. We have now established lymphoma lines of null cells, B-cells, and T-cells. Lymphoma development is preceded by the secretion into the bloodstream of large amounts of murine leukemia virus M.W. 70,000 glycoprotein antigen; however, very little virus is produced. In vivo, the expression of viral envelope antigen appears within a few days after human tumor transplantation and precedes the development of lymphoma by about a month. Cells expressing viral antigens are first seen in the diffuse cortex of lymph nodes and the periarteriolar white sheath of the spleen, the tissue domains in which lymphomas also first appear.

Effect of 4'-doxorubicin analogs on heterotransplantation of human tumors in congenitally athymic mice.

The antitumor activity of three new doxorubicin (DX) derivatives with less cardiotoxicity than the parent compound was tested against several human tumors representative of some of the major classes of human cancer. The tested DX derivatives, modified on the 4' position of the amino sugar, were 4'-epiDX, 4'-deoxyDX, and 4'-O-methylDX. Fourteen human tumors (three breast tumors, three lung tumors, three melanomas, two ovarian tumors, one prostate tumor, one sarcoma, and one larynx tumor) serially transplanted in athymic mice were used to screen the antineoplastic activity of the 4'-DX derivatives. BALB/c nude mice were treated iv with equitoxic doses of each as a single agent (less than or equal to LD10) on a weekly basis for 3-4 weeks, starting when the tumor became relatively large. 4'-EpiDX, which has a higher threshold limit of cardiac toxicity in man, was found active against breast, lung (epidermoid and oat cell carcinoma), prostate, and ovarian tumors. This drug showed particularly good activity against melanomas. 4'-DeoxyDX was active against breast and prostate tumors, while 4'-0-methylDX was active against breast and ovarian tumors and possibly sarcoma.

Isolation and characterization of plasma membranes from transplantable human astrocytoma, oat cell carcinoma, and melanomas.

Purified plasma membranes were obtained from five transplantable human tumors, a grade IV astrocytoma, an oat cell carcinoma, and three melanomas. Plasma membrane fractions were isolated from tumor homogenates by differential and discontinuous sucrose gradient centrifugation. Determination of enzyme activities indicated that the plasma membranes were enriched 10- to 20-fold with respect to 5'-nucleotidase, nicotinamide adenine dinucleotide glycohydrolase, Mg2+-activated nucleoside triphosphatase, and sialic acid. Specific activities of nearly all the enzymes varied with the individual tumors, even among tumors of the same type, i.e., the melanomas. Electron micrographs of the plasma membrane fractions showed smooth single-membrane vesicles with slight contamination by lysosomes. Therefore, these membranes are suitable for comparative biochemical studies and for the preparation of tumor-specific monoclonal antibodies. Plasma membranes from all five tumors contained very high Mg2+-adenosine triphosphatase (ATPase) activities. The Na+-K+-ATPase was a minor component of the total ATPase of these membranes (less than 30%). The major component was an ATPase exhibiting similar activity toward several nucleoside triphosphates. The activity of such a nucleoside triphosphatase has been correlated with tumorigenicity in cultured liver epithelial cells. The nucleoside triphosphatase of the plasma membranes of astrocytoma and oat cell carcinoma was stimulated from 50 to 1005 by concanavalin A, whereas ATPase of the melanoma plasma membranes was not or only slightly stimulated. The different response to concanavalin A could be due to differences in the ATPase molecules of the individual tumors or to the different environment of the ATPase.