Cellular origin and induction of pregnancy-associated alpha 2-glycoprotein synthesis in pregnant rats.

The synthesis of alpha 2-PAG was measured and compared in tissues and cells from normal non-pregnant females, and maternal and fetal rats in vitro to define the target cells hormonally regulated during pregnancy. Synthesis was measured by [L-14C]leucine incorporation into immunochemically isolated alpha 2-PAG and confirmed by radioimmunodiffusion. alpha 2-PAG synthesis was demonstrated in maternal peripheral blood leucocytes, placenta, breast, spleen, liver and fetal peripheral blood leucocytes and liver. Maternal and fetal liver were the most active tissue producers and fetal liver synthesized 4 times more alpha 2-PAG than did maternal liver. Furthermore, fetal peripheral blood leucocytes synthesized 2 times more alpha 2-PAG per cell than did these same maternal cells. A direct comparison of synthesis by cells from pregnant and non-pregnant female rats revealed that (1) maternal peripheral blood leucocytes synthesized 5 times more alpha 2-PAG per cell than did normal leucocytes, although these same cells synthesized approximately equal amounts of total cell protein per cell, (2) maternal peritoneal exudate macrophages also synthesized 5 times more alpha 2-PAG per cell than did macrophages obtained from normal female rats, and total protein synthesis by these cells also closely paralleled each other, (3) maternal and fetal plastic-adherent peripheral blood monocytes synthesized 22 and 58 times more alpha 2-PAG per cell respectively than did normal monocytes, (4) maternal and fetal non-adherent lymphocytes synthesized 8 and 16 times more alpha 2-PAG per cell respectively than did normal lymphocytes and (5) fetal monocytes and lymphocytes synthesized 3 and 2 times more alpha 2-PAG per cell than did maternal monocytes and lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo cytotoxic activity of anti-human leukemia monoclonal antibodies SN5c and SN6 daunorubicin conjugates.

Murine monoclonal antibodies SN5c specific for the common acute lymphoblastic leukemia antigen (CALLA) and SN6 specific for a novel GP160 tumor associated antigen expressed on non-T ALL and myelomonocytic leukemia cells were conjugated to daunorubicin via an intermediate dextran carrier. The resulting monoclonal antibody-daunorubicin conjugates retained the immunoreactivity of the unlabeled antibody to antigen positive leukemia target cells. In addition, these conjugates demonstrated selective cytotoxic activity when tested against a panel of human leukemia cell lines and/or human leukemia patient samples of peripheral blood or bone marrow origin. The SN5c and SN6-daunorubicin immunoconjugates were superior to a non-specific isotype matched MOPC-daunorubicin conjugate in in vitro cytotoxicity assays. Free daunorubicin, however, was more cytotoxic than either immunoconjugate but lacked selectivity. SN5c-daunorubicin and SN6-daunorubicin combined were as effective as free daunorubicin when used for in vivo therapy and led to complete ablation of established NALM-6 tumors in an athymic nude mouse model. The SN5c-daunorubicin conjugate was also shown to be significantly less toxic than free daunorubicin in non-tumor bearing Balb/c mice. These studies indicate that mAb-daunorubicin conjugates can be constructed which retain specific binding and exhibit selective cytotoxicity against human leukemia cells and suggest that they may have therapeutic applications.

Specific cytoplasmic alpha-fetoprotein binding protein in MCF-7 human breast cancer cells and primary breast cancer tissue.

Direct evidence was obtained for the existence of a specific high affinity alpha-fetoprotein (AFP)-binding protein in the cytosol of both MCF-7 human breast cancer cultured cells and primary breast cancer tissue from postmenopausal women using a nitrocellulose blotting assay. Scatchard analysis of the binding data for MCF-7 cells at 37 degrees C revealed the presence of a single class of AFP binding sites with an apparent Kd of 4.5 x 10(-8) M, and 75,000 binding sites per cell. All 9 primary breast cancer cytosols obtained from postmenopausal women also contained measureable levels of this specific AFP-binding protein. The number of AFP molecules specifically bound varied considerably between patients and ranged from 29-250 fmol per mg cytosol protein. Levels of AFP-binding protein levels and estrogen receptor measured in these same breast cancer cytosols showed a positive statistical correlation (r = 0.85). Taken together, the present evidence for the existence of a specific cytoplasmic AFP-binding protein in MCF-7 cells and previously reported evidence for de novo synthesis of free immunoreactive and bound nonimmunoreactive forms of cytoplasmic AFP by MCF-7 cells is consistent with the conclusion that most of the endogenous AFP synthesized in breast cancer cells is rapidly bound to specific cytoplasmic AFP-receptors, and that binding of AFP to these receptors masks its immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated serum alpha fetoprotein levels in postmenopausal women with primary breast carcinoma.

Elevated serum alpha fetoprotein (AFP) levels were detected and measured by a RIA in postmenopausal women with primary breast carcinoma. This increased AFP consisted of both free and total AFP measured before and after treatment of serum with 0.4 M KCl. The difference between free and total serum AFP in breast cancer patients was 44 IU ml-1. We conclude that serum from breast cancer patients contains appreciable amounts of previously undetected, initially nonimmunoreactive, AFP which became immunoreactive and measurable after KCl treatment. Previous reports indicating the presence of free and nonimmunoreactive AFP in primary breast cancer tissue cytosol and synthesis of these AFP components by MCF-7 cultured human breast cancer cells supports the conclusion that elevated serum AFP measured in breast cancer patients originates from the breast cancer cells. The conclusion that AFP is a breast tumour marker protein suggests that: (1) serial serum AFP measurements in breast cancer patients may provide useful diagnostic and prognostic information in individual cases and (2) a similar nonimmunoreactive serum AFP may be detected in patients with other types of cancer not currently known to be associated with AFP.

Biosynthesis of alpha fetoprotein by MCF-7 human breast cancer cells.

Direct evidence was obtained for de novo synthesis of AFP by MCF-7 human breast cancer cells per se. Synthesis was demonstrated by L-14C-leucine and L-35S-methionine incorporation into immunochemically isolated AFP, and confirmed by radioimmunodiffusion and radioimmunoelectrophoresis. This information indicates that AFP synthesis is associated with normal and neoplastic cells of several different histotypes, and suggests that AFP detected and measured previously in primary human breast cancer tissue cytosol (Sarcione et al., 1983) also resulted from in situ biosynthesis by breast cancer cells per se rather than uptake of exogenous AFP originating from extracellular sources. Evidence that AFP obtained after treatment of 14C-leucine radiolabelled MCF-7 breast cancer cell protein with 0.4 M KCl contained 2.6 times more radioactivity than did AFP obtained before such salt treatment is interpreted as indicating that two different molecular species of de novo synthesized AFP existed in breast cancer cells: (1) larger amount of non-immunoreactive AFP which became immunoreactive and measurable after KCl treatment, and (2) smaller amounts of free immunoreactive AFP. 14C-radiolabelled AFP obtained before and after treatment of cell protein with 0.4 M KCl codiffused, comigrated with alpha 1 electrophoretic mobility and gave an identical radioimmunologic reaction both with each other and with added carrier human cord serum AFP. Furthermore, preliminary studies indicated that radiolabelled non-immunoreactive AFP could be separated from lower-molecular-weight free AFP by chromatography on Sephadex G-200. Taken together, these findings suggest that synthesized free AFP was bound as a non-immunoreactive high-molecular-weight macromolecular complex rather than being covalently linked. Our current working hypothesis is that most of the de novo synthesized endogenous AFP in MCF-7 human breast cancer cells was rapidly and reversibly bound by hydrophobic bonding to a specific cytoplasmic AFP-receptor.

Detection and measurement of alpha-fetoprotein in human breast cancer cytosol after treatment with 0.4 M potassium chloride.

The mean concentration of total alpha-fetoprotein (AFP) measured in 51 primary breast cancer cytosols from postmenopausal women by radioimmunoassay after treatment with 0.4 M KCl was 87.2 IU AFP per ml. Little or no AFP was measured in these same cytosols before KCl treatment. After labeling KCl-treated human breast cancer cytosols with 125I, double immunodiffusion, immunoelectrophoresis, and autoradiography revealed that radiolabeled AFP codiffused and comigrated with alpha 1 electrophoretic mobility in a manner identical to that of added carrier cord serum AFP. These results indicate that human breast cancer cytosol contains appreciable amounts of a previously undetected AFP component that was initially nonimmunoreactive but became immunoreactive and measurable after KCl treatment. No positive correlation was found between the initial estrogen receptor content and AFP concentrations measured either before or after KCl treatment of breast cancer cytosols.

Demonstration of pregnancy-associated alpha 2-glycoprotein (alpha 2-PAG) in pregnant, estrogen-treated and breast cancer-bearing rats.

Three pregnancy-associated proteins were demonstrated immunologically in the sera of pregnant rats. Two of these proteins were identified as alpha-fetoprotein and alpha-macrofetoprotein. The third high molecular weight (382,000 daltons) pregnancy-associated alpha 2-glycoprotein (alpha 2-PAG) was detected in the sera of pregnant, estrogen-treated and breast cancer-bearing female rats, and gave immunologic reactions of identity. The serum concentration of this alpha 2-glycoprotein increased rapidly during pregnancy and reached a maximum during the third trimester of gestation, then decreased to trace levels within 3 days postpartum. Administration of estrogen to normal adult female rats resulted in markedly elevated serum levels of this alpha 2-glycoprotein. These animal data indicate that (1) pregnancy-associated alpha 2-glycoprotein detected in pregnant, breast-cancer bearing and estrogen-treated rats in analogous to its human counterpart, and (2) this animal model system is suitable to study sites of synthesis, and the biologic function of this pregnancy-associated protein.

Increased ferritin synthesis and release by Hodgkin's disease peripheral blood lymphocytes.

Peripheral blood lymphocytes from patients with all stages of untreated Hodgkin's disease and from normal healthy adults were shown to synthesize and release ferritin in vitro. Ferritin synthesis was confirmed by immunoelectrophoresis, double immunodiffusion and autoradiography. Hodgkin's disease lymphocytes synthesized ferritin 4.2 times faster and released it 2.4 times faster than did normal lymphocytes, whereas total protein synthesis was faster in normal lymphocytes. Patients with nodular sclerosis and perhaps those with absence of fever had the highest synthetic rates; however no relationship was observed between relative rates of lymphocyte ferritin synthesis and sex, age, anatomical stage and presence of splenic or hepatic involvement by tumor. Addition of iron to normal human lymphocytes produced little or no change in ferritin synthesis. These data indicate that part of the intracellular ferritin detected in peripheral blood lymphocytes from patients with Hodgkin's disease and from normal individuals resulted from de novo synthesis rather than from uptake and storage of serum ferritin, and suggests that elevated ferritin levels detected in the serum and tumor tissue of Hodgkin's disease patients originate from lymphocytes.

Intracellular synthesis of alpha-fetoprotein and fibrinogen without secretion by Zajdela rat ascites hepatoma cells.

Transplabtable Zajdela rat ascites hepatoma cells, previously considered "nonproducers," synthesize detectable amounts of intracellular alpha-fetoprotein (AFP) and fibrinogen, but fail to secret or release these serum proteins. Evidence for defective secretory mechanisms for serum proteins in these hepatoma cells (a) explains the failure to detect AFP in either the serum or ascitic fluid of rats bearing this hepatoma, (b) indicates that some hepatoma cells should be classified as "nonsecretors," rather than nonproducers of AFP, and (c) suggests that failure to detect AFP in some human and animal hepatomas in vivo and in vitro may also reflect failure of secretion rather than failure of intracellular synthesis.

Ferritin synthesis by splenic tumor tissue of Hodgkin's disease.

Increased ferritin synthesis by Hodgkin's disease splenic tumor tissue was demonstrated by incorporation of 14C-leucine and radioautography. This suggests that elevated tumor and serum ferritin concentrations found in patients with Hodgkin's disease is derived from tumor tissue per se.