Evaluation of oncofetal protein-related mRNA transport activity as a potential early cancer marker in dogs with malignant neoplasms.

A 55-kd protein with mRNA transport activity found in fetal rat liver cells and plasma from mice, rats, and human beings with malignant neoplasms has been designated oncofetal protein 55 (OFP55). Monoclonal antibody produced to rat OFP55 cross-reacts with human OFP55. Using this monoclonal antibody in a bioassay measuring mRNA transport stimulated by OFP55, we tested the plasma from 19 dogs with a variety of malignant neoplasms, including carcinomas, sarcomas, lymphomas, and melanomas, and compared the results with plasma from 20 clinically normal dogs without evidence of neoplasia. The mean mRNA transport activity from the group of dogs with malignant neoplasms was 0.43 +/- 0.28%/mg of protein. Mean transport activity from the group of control dogs was 0.04 +/- 0.02%/mg of protein. These means were significantly different (P < 0.0001). The degree of overlap between these 2 groups in their OFP55-related mRNA transport activity was minimal, and measurement of this protein appears to have potential for the early detection of malignant neoplasms in dogs.

Development and use of monoclonal antibodies against an oncofetal protein associated with carcinogenesis and tumorigenesis.

An oncofetal protein (OFP) studied in our laboratory associated with embryogenesis, carcinogenesis and tumorigenesis has as its known biological function the modification of RNA release from isolated nuclei. In the present study, we have developed and investigated the use of monoclonal antibodies against OFP. Six hybridoma cell lines (A-F) were isolated by screening the hybridoma culture media for anti-OFP antibodies (MOFP) with an indirect ELISA and by testing the ability of these antibodies complexed with anti-mouse IgG-agarose to bind to rat OFP and remove its associated RNA transport activity from solution (Immunobioassay). An inhibition ELISA developed to measure OFP gave a linear response up to 20 ng of plasma protein from a tumor-bearing rat. Western blot analysis using these monoclonals showed that OFP from a rat tumor (H7777) cytosol that shed to the blood consisted of two species exhibiting molecular weights of 50 and 55 kD respectively. In order to show the usefulness of our assays, a preliminary study showing the ability of the immunobioassay to detect the expression of OFP in the plasma of carcinogen treated rats in a dosage dependent manner has been presented. Since OFP is produced in the target organ of rats shortly after treatment with carcinogens and persists in the preneoplastic foci and subsequent tumors, these monoclonal antibodies will be valuable in studying its involvement in chemical carcinogenesis and tumorigenesis.

Expression of an oncofetal protein (OFP) in rat and human leukemia cells.

A unique oncofetal protein (OFP) previously identified in rat fetal tissue and rat and human solid tumors, is now shown to be present in rat and human leukemia cells by use of a monoclonal antibody-based assay. Using a highly specific anti-rat OFP monoclonal antibody OFP has been unquivocally immunolocalized to the cytoplasm of the rat leukemia cells. The factor is rapidly released to the circulation as 50 and 55 kD species which share the immunological determinants. When leukemia cells are transplanted to normal rats, OFP increases in the circulation in a biphasic manner which may be due to immune clearance since circulating anti-OFP antibodies have been demonstrated. Induction of differentiation in the human HL-60 leukemia cell line by 13-cis-retinoic acid caused a down regulation of OFP synthesis, both intra- and extra-cellular levels dropping to essentially zero. Induction of differentiation with dibutyryl cyclic AMP caused a cessation of secretion of OFP, with a marked increase in its intracellular concentration, a condition resembling the retention in fetal cells. Leukemia cells add to a growing list of tumors previously shown to produce OFP, suggesting that OFP is intimately involved in some facet of tumorigenesis.

A carcinogenesis- and tumorigenesis-associated rat fetal protein: an immuno-histochemical and immuno-biochemical study utilizing a new monoclonal antibody, MOFP.

An oncofetal protein (OFP), which is a potential marker for carcinogenesis and tumorigenesis, was evaluated with monoclonal antibodies shown to be specific for the antigen. Treatment of partially hepatectomized rats with a single non-necrogenic dose of diethylnitrosamine induced OFP in the liver. Its concentration, as measured by a dual immuno/bioassay, increased steadily over a 5-week period of observation before reaching a constant level. Immunohistochemical localization of OFP in liver sections from rats treated with N-nitroso-N-diethyl-nitrosamine showed that the factor was primarily localized to the cell cytoplasm in cells of most of the altered hepatic foci although some of this shedding antigen was also extracellular. Monoclonal antibody 17-1A specific for 17-1A antigen, an established surface marker for adenocarcinomas of the gastrointestinal tract, showed a similar distribution in liver from the carcinogen-treated rats, but localized to the cell membrane and cytoplasm. Scattered cells surrounding the altered hepatic foci were also positive for both monoclonal antibodies. Immunolocalization studies showed fetal rat liver and hepatoma were positive for OFP but adult normal or regenerating liver was negative. It was not detected in cells which morphologically could be classified as oval cells. As assessed by immuno/bioassay, the OFP released to the peripheral blood (plasma) of hepato-carcinogen-treated rats increased for 3 weeks, before undergoing a transitory decrease. Circulating antibodies specific for the factor were detected in the blood around 3-5 weeks post-treatment. Development of Western blots of the OFP with antiphosphotyrosine IgG indicates that the marker protein contains phosphotyrosine.

Developmental changes in expression of a tumor-associated protein in the rat fetus.

Monoclonal antibodies specific for a rat tumor-associated protein cross-react with a similar protein present in the cytosol of the rat fetus. The oncofetal protein exists as two species of approximate molecular weight 50 and 55 kDa which promote the transport of RNA from isolated nuclei. During rat fetal development, the protein first increases in concentration from approximately 12 to 16 days gestation and then drops to non-detectable levels perinatally.

Induction of an oncofetal marker protein upon X-radiation of rat mammary glands.

Fifty-day-old female Sprague--Dawley rats were given carcinogenic and sub-carcinogenic doses of X-radiation to the mammary glands to evaluate the induction of a 60-kDa oncofetal protein (OFP-60). This protein has previously been shown to be produced in the target organ and released to circulation during chemical carcinogenesis and tumorigenesis. In a time course experiment, the mammary glands of the rats were irradiated with a single dose of 1.85 gray (Gy). The OFP-60 marker protein was detected in peripheral blood at 21 days post-irradiation. Irradiation of the mammary gland with single X-ray doses ranging from 0.22 to 1.85 Gy produced a linear relationship between X-ray dose and plasma concentration of OFP-60 determined at 21 days post-treatment. This initiation-related parameter correlates with the known linear relationship between dose of X-radiation and potential tumor incidence.

Hybridization analysis of RNA transported from rat liver nuclei in response to 35 kDa normal and 60 kDa messenger RNA transport factors.

The transport of messenger RNA (mRNA) in response to normal adult (35 kDa) and oncofetal (60 kDa) transport factors has been studied in a reconstituted cell-free system. Poly(A)+ mRNA sequences transported by the 35 kDa and 60 kDa transport factors were compared by cDNA:RNA hybridization kinetics. Heterologous hybridization reactions indicated that a proportion of messengers transported in response to the 35 kDa factor were absent or at a markedly reduced abundance in the mRNA released by the 60 kDa factor. Recombinant DNA probes containing cDNA inserts were used to quantitate transport of rat-liver-specific alpha 2 mu-globulin and albumin mRNA from isolated nuclei in presence of the normal and tumor-specific transport factors. More alpha 2 mu-globulin and albumin messenger sequences were transported in response to the 35 kDa transport factor as compared to the 60 kDa factor. These results indicate that the 35 kDa transport protein isolated from rat liver cytosol and the 60 kDa transport protein isolated from hepatoma cytosol, differ significantly in specificity for the classes of RNA sequences released from nuclei. Monoclonal antibodies against the 60 kDa factor do not cross-react with the 35 kDa factor or other proteins as determined by the immunobioassay and by the Western blot technique.

Active transport of messenger ribonucleoprotein particles in a reconstituted cell-free system.

The ability of a reconstituted cell-free system to transport mRNA as a ribonucleoprotein particle has been examined. Poly(A) messenger ribonucleoproteins (mRNPs), UV cross-linked after release from isolated liver nuclei in a cell-free system, exhibited a buoyant density of 1.33 g/cm3 in cesium sulfate and 1.47 g/cm3 in cesium chloride, values identical to those of poly(A) mRNP isolated directly from liver polysomes. Furthermore, the in vivo and in vitro transported mRNP showed a similar degree of resistance to RNase digestion and had sedimentation coefficients approximately 2.5 times that of the isolated mRNA. Release of both total mRNA and alpha 2 mu-globulin mRNA was proportional to the concentration of a specific cytoplasmic protein. Removal of the transport proteins from the cytosol with streptomycin sulfate provided a basal system incapable of supporting the active transport of alpha 2 mu-globulin mRNA. Hybridization of released RNA with a recombinant probe specific for intron 6 of alpha 2 mu-globulin showed that intron sequences were retained within the nucleus under optimal alpha 2 mu-globulin mRNA transport conditions and that the transported alpha 2 mu-globulin mRNA was of mature size.

Analysis of nuclear RNA processing and transport by temperature perturbation of a cell-free system from mammalian cells.

The similarity of the Arrhenius plots relating temperature to messenger RNA (mRNA) transport from intact and membrane-denuded rat liver nuclei demonstrates that the ATP and cytosol-dependent transport is independent of the lipid phase of the nuclear membrane. This temperature dependence of RNA release was confirmed for alpha 2u-globulin mRNA by use of a recombinant DNA probe. Ribosomal RNA (rRNA) release showed a similar temperature dependence, suggesting that both mRNA and rRNA share a common temperature-sensitive step. The kinetics of RNA release at different temperatures suggest that RNA transport from mammalian cell nuclei is a rate-controlled rather than a graded unlocking phenomenon. The processing of mRNA precursors also exhibits a temperature dependence as shown by the linear increase in the ratio of total alpha 2u-globulin RNA to alpha 2u-globulin precursor as a function of time at 30 degrees C but not at 14 degrees C in spite of residual transport at the lower temperature. This temperature dependence of mRNA processing was confirmed by Northern blot analysis of the nuclear RNA following a 45 min incubation. Thus, both the processing and transport of RNA show temperature-sensitive steps when analyzed in cell-free systems derived from mammalian cells.

Endogenous and exogenous factors affecting ribosomal RNA release from rat liver nuclei in a cell-free system.

rRNA release from isolated liver nuclei has been analyzed in a reconstituted cell-free system using density-gradient analysis and hybridization to a specific recombinant DNA probe to monitor the process. The cell-free system was shown previously to be energy- and cytosol-dependent and to support the formation and release of functional ribosomal subunits. The release of rRNA is now shown to have an absolute dependence on a 70000 dalton cytosol protein. Although in vivo studies suggest that chronic administration of thioacetamide may block formation of a protein involved in the nucleocytoplasmic transfer of ribosomes, the 70000 dalton transport factor is not affected by the treatment. Rather the defect appears to be localized to the nucleus, since it cannot be reversed with normal cytosol from a homologous source. Early stages of nRNA processing appear to be affected by thioacetamide, although additional effects on transport are not ruled out.

Nucleocytoplasmic release of repetitive DNA transcripts in carcinogenesis correlates with a 60 kilodalton cytoplasmic protein.

Treatment of rats with the hepatocarcinogen 3'-methyl-4-dimethylamino-azobenzene (3'-MeDAB) causes the appearance in the liver cytosol of a 60 kilodalton oncofetal protein. The appearance of this factor occurs within 40 h of treatment and coincides with the increase in the amount of rapidly labeled RNA released from nuclei in a reconstituted cell-free system. Cross-over experiments show that this increase is due to an enhanced transport capacity of the cytosol. The 60 kilodalton RNA transport factor is also present in the cytosol of tumor cells. Addition of the 60 kilodalton factor to normal liver cytosol causes the transport of repetitive RNA sequences similar to those transported from liver nuclei to tumor cell cytosol and those transported to the tumor cell cytoplasm in vivo. This factor modifies nuclear RNA restriction, at least in part, by eliciting the transport of repetitive RNA normally retained within the nucleus of the normal cell.

Absence of the cancer-associated factor with a molecular weight of 60,000 from the plasma of patients with a spectrum of nonneoplastic conditions.

We demonstrated previously (Cancer Res., 42: 4964-4969, 1982) that a tumor-associated factor was consistently present in the plasma of over 100 human cancer patients with tumors at 31 different sites. The plasma of healthy controls had very low activity in the biochemical assay. In the present study, we show by a combination of molecular sieving and assay of nuclear RNA transport that the tumor-associated factor, which has a molecular weight of 60,000, is undetectable in the plasma of healthy adults. The low activity reported earlier is due to three normal cell factors of markedly different molecular weight. Furthermore, the tumor factor is shown to be absent from the plasma of male and female patients hospitalized for a variety of nonmalignant surgical conditions. Only the plasma from patients who were pregnant, suffered from chronic renal failure, or had recent myocardial infarction gave false positives in the biochemical assay. However, in these cases, the activity was due to an increase in the normal tissue-associated factors and not to the appearance of the Mr 60,000 tumor-associated factor. The factor is present in amniotic fluid, confirming that it is a fetal factor which does not cross the placental barrier. Thus, it may be classified as an oncofetal factor. All four factors found in the plasma were identified in the cytosol from a human tumor. In summary, the tumor-associated factor appears to be tumor specific and can be unambiguously identified by bioassay of the plasma factors eluting from Sepharose CL-6 B columns in the Mr 60,000 region. It can also be identified by examination of sodium dodecyl sulfate:polyacrylamide gel electrophoretograms of the appropriate Sepharose CL-6 B fractions after removal of albumin.

An oncofetal 60-kilodalton protein in the plasma of tumor-bearing and carcinogen-treated rats.

A Mr 60,000 protein, detected by its ability to induce the release of RNA from isolated nuclei, is present in the plasma of tumor-bearing and carcinogen-treated rats, together with low amounts of 2 messenger RNA transport proteins identified earlier in normal cells. The Mr 60,000 protein has been identified in tumor cell cytoplasm and in amniotic fluid, but does not appear to cross the placental barrier. Significant amounts of the Mr 60,000 oncofetal protein appear in the plasma of carcinogen-treated rats within a few weeks of treatment. It may be the fetal form of the adult messenger RNA transport proteins.

Organ and species specificity of the messenger RNA transport factor.

The transport of messenger RNA from isolated rat liver nuclei is dependent on a 35,000 dalton protein localized in the cytoplasm. The tissue and species specificity of this protein are reported. The factor from female rat liver or from rat brain, kidney, or hepatoma supports the transport of messenger RNA from male rat liver nuclei. Messenger RNA is also transported in response to the factor from beef and chicken liver. The cytosols from rat erythrocytes, amoeba and yeast were inactive in the rat liver system. The results indicate that the messenger RNA transport factor is not specific for the nucleotide sequence of the coding portion of the messenger RNA and that it is not organ or species specific within the vertebrates.

Effect of physiological concentrations of insulin and antidiabetic drugs on RNA release from isolated liver nuclei.

The addition of 10(-11) M insulin to a cell-free system from rat liver promotes the release of messengerlike RNA from isolated prelabeled nuclei. The stimulation was similar whether the nuclei were preincubated with insulin, or if insulin was added directly to the cell-free system with or without a protease inhibitor. Dot blot hybridization using cloned cDNA for alpha 2u-globulin mRNA showed that this was one of the messages whose release was enhanced by insulin. Nuclei isolated from rats treated with either of the antidiabetics tolbutamide or tolazamide showed no increase in RNA release in the presence of insulin over the concentration range 10(-5) - 10(-14) M. Furthermore, these nuclei did not release detectable levels of alpha 2u-globulin mRNA.

Poly(adenylate) polymerase activity of rat brain.

The poly(adenylate)[poly(A)] polymerase of rat brain, as in rat liver, is located primarily in the nuclear sap when nuclei are prepared under hypertonic conditions. The enzyme can be released from nuclei in two forms. Form I is prepared by gentle incubation of nuclei at 0 degrees C in hypotonic buffer. It has a Mn optimum of 0.6 mM and a pH optimum between 8 and 9. The ATP concentration curve plateaus at 0.2 mM. The optimal poly(A) primer concentration is 600 micrograms/ml, which is three times higher than that for the enzyme similarly prepared from liver. The time course of the reaction for the form I enzyme is increasing over the first 40 min and becomes nearly linear thereafter. Form I is not stimulated by either calcium or cyclic nucleotides, but is inhibited by polyamines, pyrophosphate, and high concentrations of GTP. Form II enzyme is prepared by homogenization of nuclei in hypotonic buffer. It has the same ATP and poly(A) optima as the form I enzyme but displays linear kinetics over a 60-min time course. It is slightly stimulated by cGMP and cAMP and strongly inhibited by spermine, sodium pyrophosphate, and high concentrations of GTP.

Transport of functional messenger RNA from liver nuclei in a reconstituted cell-free system.

The reliability of a reconstituted cell-free system for messenger RNA processing and transport, consisting of isolated nuclei in fortified cytosol, has been evaluated in terms of the functionality and regulated release of the transported product. The poly(A) messenger RNA transport in vitro formed appropriate initiation complexes with ribosomes in an optimized translation system and had template activity comparable to that transported in vivo. The intra-nuclear origin of this messenger RNA is supported by pulse-labeling studies, its transport from detergent-treated nuclei and the absence of the release under non-transport conditions. Serum albumin was identified by immunoprecipitation and electrophoresis as one of the products synthesized when the transported RNA was translated in vitro. The transport of messenger RNA in the cell-free system was dependent on specific cytosol (soluble cytoplasmic) proteins. These proteins, which constitutes less than 0.1% of the total cytosol proteins, are precipitated wtih streptomycin with high specificity.