Effects of acivicin and dipyridamole on hepatoma 3924A cells.

Dipyridamole inhibited the incorporation of cytidine, thymidine, uridine, and guanosine in rat hepatoma 3924A cells with 50% inhibitory concentrations of 0.2 to 0.5 microM. For deoxycytidine, the 50% inhibitory concentration was about 100 times higher (23.8 microM). Addition of a combination of cytidine, deoxycytidine, and guanosine, at an optimal concentration of 80 microM each, protected the hepatoma cells from the growth-inhibitory action of the antiglutamine drug, acivicin. The protection provided by the nucleosides was blocked by dipyridamole (6 microM), but not by nitrobenzylthionosine (30 microM). The effect on cell survival of graded concentrations of 0.25 to 1.75 microM acivicin plus dipyridamole (5 microM) and 80 microM concentrations each of cytidine, deoxycytidine, and guanosine was investigated. At an acivicin concentration of 1.75 microM, survivals in the different groups were: (a) acivicin alone, 1%; (b) acivicin plus dipyridamole, 1%; (c) acivicin plus nucleosides, 78%; and (d) acivicin plus nucleosides plus dipyridamole, 3%. Acivicin and dipyridamole were cytotoxic for hepatoma 3924A cells with 50% inhibitory concentrations of 0.5 and 20.3 microM, respectively, as measured by clonogenic assay.

Amphotericin B: a biological response modifier in targeting against the salvage pathways.

Dipyridamole, a nucleoside transport inhibitor which blocks the rescue effect of exogenous nucleosides, is a compound of interest for use in combination with antimetabolites of de novo purine and pyrimidine biosynthesis. This study has provided evidence that the dipyridamole inhibitory effect on nucleoside incorporation varied markedly during the course of cell growth in culture. Human colon cancer HT-29 cells in lag and log phases were highly sensitive to the blocking action of dipyridamole on thymidine incorporation with IC50 values of 0.06 and 0.07 microM respectively. In contrast, stationary phase cells were comparatively insensitive to dipyridamole with an IC50 of 46 microM. Amphotericin B restored the sensitivity of stationary phase cells to dipyridamole, lowering the IC50 value for thymidine incorporation to 0.03 microM. A similar pattern was observed for cytidine incorporation. Amphotericin B also enhanced the growth inhibitory action of dipyridamole in stationary phase cells. The combination of amphotericin B and dipyridamole should be potentially useful in cancer chemotherapy.

Potentiation of antimetabolite antitumor activity in vivo by dipyridamole and amphotericin B.

Previous studies have shown that dipyridamole (DP), a potent nucleoside transport inhibitor blocking the rescue effect of exogenous nucleosides, markedly potentiates the cytotoxicity of antimetabolites. However, no enhancement of the chemotherapeutic effect of antimetabolites by DP in vivo has yet been reported. This study provided evidence that the combination of DP and amphotericin B (AmB) significantly potentiated the inhibitory effect of 5-fluorouracil (FU) or methotrexate (MTX) against a panel of transplantable tumors including sarcoma 180, cervical carcinoma U14, and Lewis lung carcinoma in mice. No significant increase in toxicity was induced by this combination in treated mice. Our results indicate that the combination of DP and AmB with antimetabolites is potentially useful in cancer chemotherapy.

Mode of action of C-1027, a new macromolecular antitumor antibiotic with highly potent cytotoxicity, on human hepatoma BEL-7402 cells.

C-1027, a new macromolecular peptide antitumor antibiotic produced by a Streptomyces strain, was extremely cytotoxic to cultured cancer cells and markedly inhibited the growth of transplantable tumors in mice. As determined by tritium-labeled precursor-incorporation assay, C-1027 strongly inhibited DNA and RNA synthesis in hepatoma BEL-7402 cells without affecting protein synthesis. After incubation with the hepatoma cells for 4 h, IC50 values for [3H]-thymidine and [3H]-uridine incorporation were 0.00012 and 0.00032 microM, respectively. After 30 min incubation, C-1027 showed much stronger inhibition of [3H]-thymidine incorporation than did Adriamycin, mitomycin C or methotrexate, even at a concentration 10,000 times lower. The effect of C-1027 on pBR322 DNA suggested that the drug could cause single- or double-strand scission of DNA. As determined by flow cytometry, C-1027 delayed the progression of hepatoma cells through the S-phase and blocked the cells at G2+M. Cytological study showed that C-1027 caused a drastic reduction of the mitotic index within 1 h and that an overshot of the mitotic index occurred at 48 h. Our results indicate that C-1027 is an interesting compound with highly potent activity on cellular DNA.

Salvage pathways as targets of chemotherapy.

This paper discussed the significance of the activities of purine and pyrimidine salvage enzymes in cancer cells and the targeting against them of chemotherapy. 1. The activities of salvage enzymes in the rat liver were orders of magnitude higher than those of the rate-limiting enzymes of de novo biosynthesis. A similar relationship was observed in rat hepatomas of different growth rates and in primary colon carcinoma in human. 2. The concentrations of nucleosides and nucleobases were measured in plasma, liver and hepatoma 3924A in the rat. The freeze-clamp method was required to determine the concentrations of these precursors in rat liver and hepatoma in a reliable and precise fashion because ischemia markedly altered the concentrations of nucleosides, nucleobases and, as shown earlier, nucleotides in these tissues. The results indicated that the liver markedly concentrated the purine precursors, hypoxanthine, guanine and adenine, but not thymidine, which was one-third that of the plasma. Uridine and deoxycytidine occurred in the same concentration as in plasma, but cytidine was 3-fold higher in liver. In the hepatoma in comparison to the liver the concentrations of the nucleosides and bases were altered and for some of the changes the enzymic differences between liver and hepatoma appeared to be accountable. 3. Kinetic parameters for purine and pyrimidine synthetic enzymes and for the substrates and co-factors were determined in liver and hepatoma 3924A. When enzymic activities were calculated at the tissue steady-state concentrations of the various ligands, the activities of the salvage enzymes were markedly higher than those of the rate-limiting enzymes. 4. Hepatoma cells were highly sensitive to the action of the transport inhibitor, dipyridamole, in lag and log phases. However, plateau phase cells lost their sensitivity to dipyridamole. 5. Amphotericin B rendered plateau phase cells sensitive to the inhibitory action of dipyridamole for the incorporation of thymidine. 6. Amphotericin B enhanced cytotoxicity of dipyridamole in hepatoma and human colon cancer HT-29 cells. 7. In these studies we discovered the decreased responsiveness to dipyridamole of plateau phase cells and the ability of amphotericin B to restore the sensitivity. Moreover, dipyridamole and amphotericin B were synergistic in their cytotoxic action in rat hepatoma cells and human colon cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Multi-enzyme-targeted chemotherapy by acivicin and actinomycin.

On the basis of our observation of the increased specific activities of glutamine-utilizing enzymes in purine and pyrimidine metabolism in hepatoma 3924A, and because the concentration of glutamine is ten times lower in the hepatomas than in the liver, the biochemical pharmacology of the anti-glutamine agent, acivicin, was examined. (1) Acivicin competitively inhibited the activities of amidophosphoribosyl-transferase, CTP synthetase and carbamoyl-phosphate synthetase II from extracts of liver and hepatoma 3924A. (2) In addition to the competitive inhibition exerted by acivicin, evidence was obtained that this drug also irreversibly inactivated in vitro the glutamine-utilizing enzymes. It is particularly relevant for the selectivity of acivicin that the activity of aspartate carbamoyltransferase, an enzyme present in the same complex as carbamoyl-phosphate synthetase II, was not affected by the anti-glutamine agent. (3) Acivicin in vivo brought down the activities of glutamine-utilizing enzymes in a period of 10 min to 1 hr after injection. CTP synthetase activity declined to less than 10% of that observed in the uninjected rats. The decreases were not reversible by various in vitro methods, but in vivo the activities returned to normal range in 72 hr. (4) The activity of aspartate carbamoyltransferase, which exists as a multi-enzyme complex with synthetase II, was not altered by acivicin injection. Similar results were observed in transplantable sarcoma in the rat. (5) The acivicin-induced decrease in enzymic activities could not be restored by purification of the enzymes. (6) In vitro studies indicated that addition of acivicin to liver or hepatoma extracts or purified enzymes rapidly decreased enzymic activities; the activities could not be restored. These results are consistent with an interpretation that acivicin acts either as a tight-binding inhibitor or as an inactivator through alkylation of the enzymes of glutamine utilization. (7) Acivicin in combination with actinomycin provided a synergistic kill of hepatoma cells in tissue culture and also inhibited the growth of transplantable solid hepatoma 3924A in the rat. (8) The synergistic biological results of combination chemotherapy with acivicin and actinomycin can be accounted for by the action of acivicin in inhibiting GMP and CTP synthetases, resulting in a decrease in GTP and CTP content, and by the actinomycin-caused inhibition of RNA polymerase in selectively blocking the utilization of GTP and CTP.

Azidothymidine and dipyridamole as biochemical response modifiers: synergism with methotrexate and 5-fluorouracil in human colon and pancreatic carcinoma cells.

Azidothymidine (AZT), inhibiting thymidine kinase (EC 2.7.1.21) (Weber, G. et al., Cancer Commun. 2:129-133, 1990) and dipyridamole, inhibiting nucleoside transport (Zhen, Y.-s. et al., Cancer Res. 43:1616-1619, 1983) exert blocking action on the activities of salvage pathways of nucleotide biosynthesis. Determined by clonogenic assay in human colon cancer HT-29 cells, the cell survivals for AZT, 10 microM, dipyridamole, 5 microM, and methotrexate (MTX), 0.025 microM, were 90, 82, and 62%, respectively; while the combinations of AZT + MTX, dipyridamole + MTX and AZT + dipyridamole + MTX, reduced survivals to 36, 4.3, and 0.7%. AZT or dipyridamole was synergistic with MTX, whereas AZT plus dipyridamole showed an even more marked potentiation of MTX activity. The survivals for 5-fluorouracil (5-FU), 0.5 microM, alone, AZT + 5-FU, dipyridamole + 5-FU, and AZT + dipyridamole + 5-FU were 86, 47, 29 and 5.1%, respectively. Similar results were observed in human pancreatic carcinoma BxPC-3 and PANC-1 cells. AZT markedly enhanced the inhibitory effect of dipyridamole in reversing the thymidine-hypoxanthine rescue from MTX cytotoxicity. AZT inhibited [14C]thymidine incorporation into DNA in HT-29 cells and strongly enhanced the effect of dipyridamole. The results indicate that combinations composed of AZT, dipyridamole, and antimetabolites, such as MTX and 5-FU, are potentially effective in the chemotherapy of human neoplasias.

A new macromolecular antitumor antibiotic, C-1027. III. Antitumor activity.

C-1027, a new macromolecular antitumor antibiotic produced by Streptomyces globisporus C-1027, showed extremely potent cytotoxicity toward cultured cancer cells. Compared in terms of IC50 values, antibiotic C-1027 showed much more potent cytotoxicity than doxorubicin, mitomycin C and neocarzinostatin. Spermatogonial assay, a prescreen for anticancer drugs, was highly sensitive for detection of C-1027. At tolerable doses, C-1027 exhibited marked inhibition on a panel of transplantable tumors in mice, which included leukemia L1210, P388, ascites hepatoma H22, sarcoma 180 and melanoma Harding-Passey.

[Antitumor activity of new antitumor antibiotic C1027 and its monoclonal antibody assembled conjugate].

C1027, a new macromolecular peptide antitumor antibiotic produced by Streptomyces globisporus C1027, shows extremely potent cytotoxicity to cultured cancer cells. The antibiotic is composed of an apoprotein and a chromophore and the latter serves as the active part of the compound. C1027 was separated into apoprotein and chromophore by methanol extraction and the separated parts can be reconstituted to form the active C1027 molecule in phosphate buffer. For determination of the specificity of C1027 reconstitution, the apoprotein was incubated with epirubicin and the chromophore was incubated with H16, a McAb directed against hepatoma cells. Notably, the reconstitution of C1027 occurred neither between apoprotein and epirubicin nor between chromophore and IgG molecule. In addition, bovine serum albumin showed no competition with C1027 apoprotein in binding to the chromophore. Various methods for linking C1027 to McAb were studied and two kinds of immunoconjugates have been prepared: (1) direct conjugate was made by linking C1027 to McAb, using SPDP as a linker agent, (2) assembled conjugate was made by linking and reconstitution, including 3 steps. Firstly, the chromophore was extracted with methanol and stored at -70 degrees C in drak. Secondly, the apoprotein was conjugated to McAb by SPDP and finally the extracted chromophore was added to the McAb-apoprotein conjugate. Determined by clonogenic assay, the IC50 values for hepatoma cells were 42 pmol/L, and 5.5 pmol/L, respectively, for direct conjugate and assembled conjugate. The IC50 value of M3-C1027 assembled conjugate prepared by linking the irrelevant McAb M3 to C1027 was 1,400 pmol/L.(ABSTRACT TRUNCATED AT 250 WORDS)

[Relationship between the molecular composition of C1027, a new macromolecular antibiotic with enediyne chromophore, and its antitumor activity].

The molecule of C1027, an antitumor antibiotic with extremely potent cytotoxicity against cultured cancer cells, is composed of an enediyne chromophore and an apoprotein of 10.5 kDa. These two parts of the molecule, connecting each other through non-covalent binding, can be dissociated and reconstituted. As determined by clonogenic assay, the chromophore served as the active part of the molecule, displaying potent cytotoxicity similar to that of intact C1027. The activity of free chromophore decreased more rapidly than that of intact C1027, indicating that apoprotein played a role in protecting the chromophore from inactivation. By incubating together in phosphate buffer, the chromophore and apoprotein were reconstituted to form an intact C1027. The ratio of chromophore and apoprotein remained 1 : 1 in the reconstituted molecule, even though extra amount of chromophore was added. The optimal condition for the reconstitution was pH 7.0, at 20 degrees C for 12 h. When the disulfide bond of the apoprotein was reduced by DTT, the activity of C1027 decreased more rapidly. C1027 was digested by pronase and the produced fragments of various molecular weights were examined by capillary electrophoresis. The cytotoxicity of 3-5 kDa fragment approximated that of intact C1027 and its IC50 value was 0.07 fmol.L-1. The results indicate that the intactness of the apoprotein is not indispensable for stabilizing the chromophore and a smaller molecule of 3-5 kDa consisting of a peptide fragment and a chromophore may retain full C1027 activity.

[Rhein induces apoptosis in cancer cells and shows synergy with mitomycin].

AIM: To study the apoptosis-inducing and growth-inhibitory effect of rhein, an herb-derived compound, and its combination with mitomycin C (MMC) on cultured tumor cells. METHODS: MTT assays were used to determine the inhibition of proliferation by drugs in several tumor cell lines. Nucleoside transport and DNA synthesis inhibition were determined by [3H] thymidine transport and incorporation assays. Flow cytometry, electrophoresis on agarose gels and morphological assessment were applied to analyze the apoptotic changes. RESULTS: The IC50 values of nucleoside transport was 19.1 micrograms.mL-1 and that of the DNA synthesis inhibited by rhein was 27.4 micrograms.mL-1. In MTT assay the IC50 values of rhein for KB, hepatoma BEL-7402 and mammary carcinoma MCF-7 cells were 11.5 micrograms.mL-1, 14.0 micrograms.mL-1 and 18.4 micrograms.mL-1 respectively. Synergistic effect of rhein and MMC was found in all the three cell lines. As found, rhein induced apoptosis in KB cells, and the increase of apoptotic cells reached 71.0% at 96 h. The combination of rhein and MMC enhanced the induction of apoptosis significantly. CONCLUSION: These results suggest that rhein, as a biochemical modulator, might be potentially useful in cancer chemotherapy.

[Experimental studies on the antitumor activity of monoclonal antibody--bleomycin A6 conjugate against human liver cancer].

Bleomycin A6 (A6), a single component of bleomycin complex, is highly active against human liver cancer cells in vitro and xenografts in nude mice. A6 was conjugated to monoclonal antibody H111 directed against human hepatoma BEL - 7402 cells, using Dextran T40 as an intermediate. The conjugate consisted of a coupling molar ratio of 1:264 for H111 and A6, and retained 6.3% of A6 activity. As determined by clonogenic assay with hepatoma BEL - 7402 cells exposed to the agents for 1 h, the IC90 values for H111 - A6 conjugate, free A6 and M3 - A6 conjugate (an irrelevant conjugate) were 0.17 mu mol/L, 17 mu mol/L and 7 mu mol/L respectively. The cytotoxicity of Hill - A6 conjugate to target cells was markedly blocked by unconjugated H111 but not by irrelevant monoclonal antibody M3. The H111 - A6 conjugate exhibited 78% inhibition on the growth of hepatoma BEL - 7402 xenografts in nude mice, whereas the equivalent doses of free A6, M3 - A6 conjugate and H111 plus A6 mixture showed approximately 30% inhibition. Histopathological examination showed no toxic changes in the liver, lung, kidney and bone marrow in the H111 - A6 conjugate--treated animals. These results suggest that the conjugate of monoclonal antibody and bleomycin A6 exhibits specific cytotoxicity to target liver cancer cells and the conjugate is highly effective against liver cancer xenografts in nude mice with more marked tumor inhibition than free A6 at comparable dose levels.

[Inhibitory effect of boanmycin on the growth of colon carcinoma 26 and hepatic metastasis in mice].

AIM: To investigate the therapeutic effect of boanmycin (BAM, bleomycin A6) on colon carcinoma 26 and its hepatic metastasis in mice. METHODS: A series of models including subcutaneous transplantation, orthotopic transplantation in cecum subserosa, intra-hepatic transplantation of tumor, and intrasplenic transplantation of tumor accompanied with hepatic metastases were employed. In addition, LEICA Q 500IW image analysis system was used to determine the area of metastatic lesions in the liver in histopathological sections. RESULTS: BAM at 5 mg.kg-1 and 2.5 mg.kg-1 inhibited the growth of subcutaneous tumors by 78.7% and 61.9%, and the orthotopic tumors by 99.4% and 90.0%; and the intra-hepatic tumors by 86.9% and 75.7%, respectively. Determined by the numbers of metastatic nodules, BAM at 10 mg.kg-1 and 5 mg.kg-1 inhibited hepatic metastases from intra-splenic transplantation by 97.6% and 56.8%; and evaluated by image analysis of metastatic lesions, by 100% and 63.3%, respectively. CONCLUSION: Boanmycin is highly effective in a panel of models including the subcutaneous, orthotopic, intra-hepatic transplanted tumors, and hepatic metastases of murine colon carcinoma 26.

Synergistic effects of geldanamycin and antitumor drugs.

AIM: To study the effect of geldanamycin (GDM) on cell-cycle of human hepatoma BEL-7402 cells and the antitumor activity of cisplatin and mitomycin C in combination with GDM in vitro and in vivo. METHODS: MTT assay was used to determine the growth inhibition of hepatoma BEL-7402 cells. Cell cycle was analyzed by flow cytometry. Transplantable murine hepatoma 22 model was used to evaluate the antitumor activity of drugs in vivo. RESULTS: The IC50 value of GDM for hepatoma BEL-7402 cells by MTT assay was found to be 0.28 mumol.L-1. At concentrations of 0.1, 1.0, and 10 mumol.L-1, GDM reduced the proportion of S phase and induced G2/M arrest in BEL-7402 cells. At relatively low cytotoxic concentration, 0.1 or 0.2 mumol.L-1, GDM markedly potentiated the cytotoxicity of a series of chemotherapeutic agents including cisplatin, mitomycin C, adriamycin and cytarabine against BEL-7402 cells. The inhibition of tumor growth by cisplatin and mitomycin C was also enhanced in transplantable hepatoma 22-bearing mice when these agents were administered in combination with GDM 0.38 mg.kg-1. The synergistic effects were very significant with CDI < 0.7. CONCLUSION: These results suggest that GDM, as a biochemical modulator targeting Hsp90 function, may be potentially useful in cancer chemotherapy.

[Minocycline potentiates the antimetastatic effect of boanmycin].

Boanmycin (bleomycin A6, BAM) was found to markedly inhibit the spontaneous pulmonary metastasis of Lewis carcinoma in mice. Compared at equitoxic doses (1/9 LD50), BAM was more effective than mitomycin. Minocycline (MNO) at 5 mg.kg-1 showed no inhibition on the growth of sc transplanted Lewis primary tumor; however, it markedly potentiated the antimetastatic effect of BAM. Treated with BAM (5 mg.kg-1) alone, the number of total metastatic foci and that of large foci (> 2 mm in diameter) in the lung were suppressed by 67% and 85%, respectively. When BAM was used in combination with MNO, the number of those foci was further reduced by 88% and 100%, respectively. By NAG enzyme assay, MNO was not cytotoxic and showed no synergism with BAM against PG cells, a cell line derived from a highly metastatic human giant cell carcinoma of the lung. Determined by ELISA with a monoclonal antibody, the expression of type IV collagenase in PG cells was remarkably inhibited by MNO. The intracellular free Ca2+ level in PG cells was reduced from 76.7 nmol.L-1 to 42.2 nmol.L-1 by MNO treatment. The study suggests that the combination of boanmycin and minocycline may be useful for control of tumor metastasis and the inhibition of type IV collagenase expression may be involved in the mechanism of minocycline potentiation.

[A fungus-derived novel nucleoside transport inhibitor potentiates the activity of antitumor drugs].

Antibiotic C3368-B (CB), identified as 3,9-dihydroxy-1-methoxy-7-methylanthraquinone, is produced by a fungus strain, Chrysosporium verrucosum Tubaki, isolated from a soil sample collected from Antarctica. CB was found to be a highly-active nucleoside transport inhibitor. By radiolabelled nucleoside assay, CB was shown to markedly inhibit thymidine and uridine transport in Ehrlich carcinoma cells, with IC50 values of 7.5 and 9.6 mumol.L-1 respectively. CB showed fairly low cytotoxicity to tumor cells. The IC50 values for epidermoid cancer KB cells and hepatoma BEL-7402 cells in clonogenic assay was 77 and 69 mumol.L-1. At relatively noncytotoxic concentrations, CB markedly enhanced the cytotoxicity of methotrexate, 5-fluorouracil, mitomycin C against KB cells and BEL-7402 cells. CB was also found to partly reverse the multi-drug resistance to vincristine and actinomycin D in leukemia L1210/MDR cells. The IC50 values were reduced by 4.9-fold (1.75 to 0.36 mumol.L-1) for vincristine and 3.3-fold (0.39 to 0.12 mumol.L-1) for actinomycin D. These results suggest that CB, as a newly-found nucleoside transport inhibitor, may be potentially useful in cancer chemotherapy.

[Antitumor effect of the immunoconjugate composed of antibiotic C1027 and Fab fragment from a monoclonal antibody directed against human hepatoma].

Antibiotic C1027, a macromolecular peptide with highly potent cytotoxicity to cultured cancer cells, was conjugated to monoclonal antibody 3A5 and its Fab fragment separately using SPDP as the linker agent. McAb 3A5, identified as IgG1, was directed against human hepatoma BEL-7402 cells. By ELISA, McAb 3A5 and the Fab fragment were strongly reactive with hepatoma cells and weakly reactive with KB cells. Determined by clonogenic assay against hepatoma BEL-7402 cells, the IC50 values for McAb-C1027, Fab-C1027 and C1027 were 4.2 x 10(-14), 8.6 x 10(-16) and 6.5 x 10(-16) mol.L-1, respectively. Fab-C1027 was 49-fold more potent than McAb-C1027 in cytotoxicity. Moreover, Fab-C1027 was 160-fold more potent in cytotoxicity to hepatoma cells than to KB cells, indicating selective cytotoxicity of Fab-C1027 conjugate to the target cells. Therapeutic effect of the conjugate was evaluated with hepatoma BEL-7402 xenograft in nude mice. After subcutaneous transplantation of the tumor, treatment started on day 3, iv, with equivalent dose of C1027, 0.1 mg.kg-1 x 6. Fab-C1027 and free C1027 inhibited tumor growth by 85% and 59%, respectively. Fab-C1027 conjugate showed more marked antitumor effect in vivo than free C1027.

[A monoclonal antibody directed against an enediyne antitumor antibiotic and its preliminary application].

C1027, composed of an enediyne chromophore and an apoprotein of 110 amino acid residues, is a new highly potent macromolecular antitumor antibiotic. In order to prepare monoclonal antibodies (McAb) against C1027 by hybridoma technique, natural C1027 was inactivated by ultraviolet and coupled with human serum albumin, then immunized BALB/c mice. After cell fusion and screening by ELISA, a hybridoma secreting anti-C1027 McAb designated as F9 was obtained. McAb F9 specifically reacted with C1027 as determined by immunoblot assay. No obvious difference was observed between the reactivity of McAb F9 to natural C1027 and that of McAb F9 to ultraviolet inactivated C1027. This result indicates that the ultraviolet-sensitive chromophore of C1027 may not participate in the epitope for McAb F9. The isotype of F9 is IgG1 and its affinity constant was found to be (2.2 +/- 0.47) x 10(7) L.mol-1 according to Beatty's method. By clonogenic assay, McAb F9 neither affected the cytotoxicity of C1027 to KB cells nor blocked the activity of the chromophore. McAb F9 also specifically reacted with the recombinant truncated C1027 apoprotein in which 16 amino acid residues at the C terminus were deleted. This study suggests that F9 is a valuable McAb for the research of C1027 apoprotein engineering, C1027 related immunoconjugates and screening of new macromolecular antitumor substances with homology to the protein part of C1027.

[Experimental studies on therapeutic effect of rat monoclonal antibody-bleomycin A6 conjugate against human colorectal cancer].

Bleomycin A6 (A6), a single component of bleomycin complex, is highly active against human colon and cecum cancer cells in vitro and xenografts in nude mice. R19, a rat monoclonal antibody against human cecum cancer Hce-8693 cells, was linked to A6. R19-A6 conjugate retained complete activity of McAb R19 and 10% activity of A6. As determined by clonogenic assay with human cecum cancer Hce-8693 cells for 1 hour exposure. The 50% inhibitory concentration (IC50) values for R19-A6, A6 and M3-A6 (conjugate of irrelevant Mc-A6) were 0.019, 1.05 and 1.00 mumol/L, respectively. The effect of the conjugate R19-A6 was 55-fold stronger than that of free A6 and 53-fold than irrelevant conjugate M3-A6. Clonogenic assay with human colon cancer HT-29 cells showed that the IC50 values were 0.078 mumol/L and 4.0 mumol/L for R19-A6 and free A6, respectively. The cytotoxicity to Hce-8693 and HT-29 cells was markedly blocked by unconjugated McAb R19 but not by irrelevant McAb MARK-3. The R19-A6 conjugate exerted 90% inhibition on the growth of cecum cancer Hce-8693 xenografts in nude mice, whereas equivalent doses of free A6, R19 plus A6 mixture and M3-A6 showed 52%, 34% and 48% inhibition, respectively. Histopathological examination showed no toxic changes in the heart, lung, liver, kidney and bone marrow in the R19-A6 conjugate treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

[Specific binding and internalization of anti-CCT2 monoclonal antibody and bleomycin A6 conjugate in human leukemia cells].

The immunoconjugate of anti-CCT2 monoclonal antibody linked to bleomycin A6 was adsorbed on colloidal gold particles (McAb-A6-Au). Binding and internalization of McAb-A6-Au particles in human leukemia CEM cells were examined by electron microscopy. After 60 min at 4 degrees C, McAb-A6-Au particles were bound to the surface membrane of 78% of CEM cells. Transferring to 37 degrees C for 15 min, McAb-A6-Au particles were found to be 56% inside the CEM cells and about one third of the cells contained particles in the nucleus. After 4 h at 37 degrees C the percentage of CEM cells containing McAb-A6-Au particles increased to 72%. However, only 14% of the antigenically irrelevant U937 cells contained these particles and none of them was found in the nucleus. Preincubation with unconjugated anti-CCT2 monoclonal antibody markedly blocked the McAb-A6-Au particle uptake in CEM cells. The McAb-A6-Au particles were internalized through the formation of endocytotic vesicles. In addition, some McAb-A6-Au particles were able to penetrate the plasma membrane directly into cytoplasma and notably into the nucleus. Results indicate that the immunoconjugate of monoclonal antibody linked to bleomycin A6 showed selective binding to target cells and entered the cells specifically and rapidly.