Transient inhibition of L929 cell mitosis and locomotion by argon ion laser irradiation.

Argon ion laser irradiation of L929 cells transiently inhibits both entry into and passage through mitosis without affecting clonogenic survival. Anaphase mitotic figures virtually disappear from irradiated cell monolayers although prophase + metaphase mitotic figures can still be identified. The total number of mitotic figures does not change significantly and time-lapse video recording shows that cells do not enter mitosis following irradiation. This effect is dependent on light dose within the 900-2700 J/cm2 range and persists for 10-48 h depending on the initial light exposure. Inhibition of cell locomotion and subsequent recovery were observed to occur over a similar time course. The possible contribution of these phenomena must be considered whenever biological systems are exposed to argon ion laser irradiation.

Mechanism of action and spectrum of cell lines sensitive to a doxorubicin-transferrin conjugate.

A transferrin-doxorubicin conjugate exhibited greatly increased cytotoxicity relative to unconjugated doxorubicin toward a variety of cultured tumor cell lines. An L929 cell line selected for doxorubicin resistance was as sensitive to the transferrin-doxorubicin conjugate as was the parental unselected line. Quantitative measurements of doxorubicin fluorescence in single L929 cells showed that uptake was similar in amount when cells were exposed to equivalent concentrations of doxorubicin presented either free or as the transferrin-doxorubicin conjugate. However, unconjugated drug fluorescence was distributed in membranes, cytoplasm and nucleus, whereas conjugate fluorescence was confined mainly to the cytoplasmic compartment. In as much as NADPH-dependent free radical formation is a known mechanism of doxorubicin cytotoxicity, localization in the vicinity of NADPH production might facilitate this cytotoxic pathway. Neither cytotoxicity nor uptake of the conjugate quantified by doxorubicin fluorescence was significantly blocked by excess free transferrin, and the conjugate was not concentrated in the plasma membrane at 4 degrees C. These findings suggest that conjugate internalization is not entirely dependent on transferrin receptor binding.

Mechanism of action and spectrum of cell types susceptible to doxorubicin photochemotherapy.

We have shown that, whereas argon ion laser irradiation alone is not cytocidal for L929 cells, it greatly increases the cytotoxicity of intracellular doxorubicin. The present study showed that light enhancement of doxorubicin cytotoxicity was not restricted to stock L929 cells, but could also be demonstrated using L929 cells selected for doxorubicin resistance and several standard cell lines that are relatively resistant to doxorubicin prior to selection. Light-enhanced cytotoxicity resulted in extensive nuclear DNA loss and was strongly inhibited by anoxia. These findings suggest that the mechanism by which light exposure enhances doxorubicin cytotoxicity involves DNA damage by intranuclear generation of reactive oxygen species.

[Tumor infiltrating lymphocytes inhibit spontaneous lung metastasis of LA795 adenocarcinoma in mice].

Tumor Infiltrating Lymphocytes (TILs) isolated from LA795 lung adenocarcinoma in mice have a remarkable antitumor activity, by using T739 syngeneic mice. TILs cytolytic activity in vitro is higher than that of LAK cells. TILs significantly inhibit spontaneous lung metastasis in vivo. The results in experiment I showed the following: TILs injection in tail vein, the inhibitory rate is 73.3%; TILs injection at tumor, 79.9%. The results in experiment II are similar to these in experiment I, and TILs antitumor activity in vivo is as three times as that of LAK cells.

[Effects of beta-carotene on lung cancer].

Beta-carotene at concentration of 6.25 micrograms/ml was shown to inhibit significantly the colony forming efficiency (CFE) of cultured human lung cancer 801 cell line and at 12.5 micrograms/ml was shown to completely inhibit CFE. A 42%-68% (P < 0.01) decrease in spontaneous lung metastasis of LA795 murine pulmonary adenocarcinoma was observed when T739 inbred strain mice were fed a diet with beta-carotene (25mg/100g diet). Ability of DNA and RNA synthesis of lung cancer cells were decreased (P < 0.05) after treating with beta-carotene (25 micrograms/ml) by 24 hours, but the ability for DNA synthesis of human lymphocytes was not effected by the same treatment. Expression of ras oncogene was proved to be inhibited by beta-carotene because the product of ras p21 proteins of cancer cells was decreased after treating by beta-carotene.

[Monoclonal antibody against human lung carcinoma].

Spleen cells of BALB/c mice immunized with human pulmonary adenocarcinoma cell LTEP-a2 were fused with murine myeloma cell SP 2/0, from which 4 hybridomas (2 A7, 2 E9, 4 F2 and 5 F11) were obtained. Indirect immunofluorescence test showed that these 4 monoclonal antibodies reacted with human lung cancer cells, but not with 2 BS or the lymphocytes and red blood cells in 4 different ABO groups of 10 persons. Using ABC immunoperoxidase stain technique, these 4 antibodies showed negative reaction with 9 tissue types from the normal subject and fetus but could react with 52-83% of the 29 human lung carcinomas and 64-92% of the 24 non-small cell lung cancers (non-SCLC). When 5 F11 was combined with 2 A7 or 2 E9, the percentage of positive stain was 100% in 24 non-SCLC. The results of indirect immunofluorescence stain showed that strong membrane stain by 5 F11 and membrane stain by 4 F2 were obtained, indicating that these antibodies could recognize antigens on cancer cell membrane. It is suggested that a mixture of 5 F11 and other antibodies be useful in the diagnosis and treatment of lung cancer. Molecular weight of the antigens recognized by the 4 antibodies was determined by SDS-PAGE and immunoblot technique to be 47 KD (2 A7), 67 KD (2E9), 40 KD (4F2) and 56 KD (5 F11).

[Antitumor effect of methylene blue in vivo].

Methylene blue can inhibit the growth of Ehrlich ascitic tumor, L1210 leukemia and P388 leukemia in mice. The average life span of the treated animals was obviously longer than that of the controls. Methylene blue was superior to 5-Fu in the treatment of L1210 leukemia of mice and two thirds of the mice with P388 leukemia were healthy and survived for more than 60 days after administrated different doses of methylene blue. When adriamycin was given simultaneously with methylene blue to mice, the acute toxic effect of adriamycin was decreased and the survival time prolonged. Methylene blue is a highly ionized and a rapidly eliminated drug which can combine well with tissues at higher concentration maintained for several hours and can easily pass through the blood-brain barrier.

[Establishment of human small cell lung carcinoma cell line (LTEP-sm 1) in nude mice and in vitro].

A serially transplatable tumor model was established by inoculating subcutaneously the surgical specimen of a patient with mixed type small cell lung carcinoma into nude mice (BALB/C nu/nu). A human small cell lung carcinoma cell line cultured in vitro was established from that tumor in nude mice. The cultured cell line gave a typical growth pattern with small round feature, cell clusters in suspension and float growth. Pathological sections of both the patient's tumor and transplantable tumor in nude mice showed the same morphological features. The typical APUD granules were observed in electron transmission microscopy. The transplantable tumor has been transferred for 9 passages in 14 months and the cell line in vitro for 71 passages in nearly one year.

Rat antikeratin monoclonal antibodies are specific for stratified squamous epithelia.

Seven monoclonal antibodies of rat origin were generated against human epidermal keratins and were assayed by immunoblot and by immunoperoxidase staining of frozen sections from various human epithelial tissues. By immunoblotting, the antibodies recognized two different subsets of high molecular weight cytokeratin polypeptides. Unlike the majority of the reported mouse monoclonal antibodies obtained to similar immunogens, these rat monoclonal antibodies showed an unexpected degree of specificity, i.e., they stained only stratified squamous epithelia and not simple epithelia nor urothelia. The potential of using rats and the appropriate immunogen to generate more tissue-specific anticytokeratin monoclonal antibodies is discussed.

Quantitation and intracellular localization of the 85K heat shock protein by using monoclonal and polyclonal antibodies.

Two monoclonal antibodies have been produced against the human 85,000-molecular-weight heat shock protein (hsp85). One of these, 16F1, cross-reacts with the murine homolog and is shown by peptide map immunoblots to be directed against an epitope different from that recognized by the other monoclonal antibody, 9D2. Both monoclonal antibodies recognize only a single Mr-85,000 species in two-dimensional immunoblots. Immunoprecipitation did not reveal an association of this heat shock protein with any other protein in HeLa cells. Immunoperoxidase staining showed a purely cytosolic distribution at both light and electron microscopic levels and no association with membranes, mitochondria, or other organelles. The 9D2 monoclonal and a polyclonal antimurine hsp85 antibody were used to identify the antigens and to quantitate their levels in a variety of normal tissues by immunoautoradiography. Relative abundance in the various tissues as determined by Coomassie blue staining correlates reasonably well with the immunoreactivity. Testis and brain, for example, have high hsp85 levels, whereas heart and skeletal muscle have little or none. The Mr-85,000 sodium dodecyl sulfate-polyacrylamide gel band in testis and brain lysates was further confirmed to be hsp85 by one-dimensional partial proteolytic peptide mapping. Based on these data and our previous observations showing that synthesis and levels of the protein are altered by depriving cultured cells of glucose, we speculate that intracellular hsp85 levels depend on differences in the intermediary metabolism of glucose in the various tissues. Furthermore, it appears that high basal levels of this heat shock protein may not necessarily protect cells against heat shock, since testis is one of the most heat-sensitive tissues and has the highest hsp85 level.