Drug sensitivity of ten human tumor cell lines compared to mouse leukemia (L1210) cells

L1210 leukemia cells, because of their rapid growth rate in suspension culture and high growth fraction, are ideally suited to screen in vitro for cytotoxic compounds. Although L1210 cells may mimic rapidly growing tumors, they have not been effective in selecting agents active against slow growing solid tumors. We expected that cell lines originating from human solid tumors, because of their slower growth rate and lower S phase fraction, would be more drug resistant than L1210. Therefore, we compared ten human tumor cell lines (5 melanomas, 4 colon carcinomas and 1 small cell lung carcinoma) to L1210 growth inhibition by 9 anti-tumor drugs. Not one human tumor cell line was consistently more resistant to all nine drugs than L1210 when the cells were exposed to drugs for about 2 doubling times. The drug sensitivity of 2 cell lines (L1210 and SK MEL 28) was again determined after a short term (2 hr) exposure and using growth inhibition and cell survival as end points. For both end points these two cell lines exhibited a random pattern of sensitivity to the drugs tested. Cell kill showed an order of sensitivity different than growth inhibition. The implication of these findings for drug-screening is discussed.     

Cytotoxicity of combinations of prostaglandin D2 (PGD2) and antitumor drugs for B16 melanoma cells in culture

Prostaglandin D₂ (PGD₂) is lethal to murine and human melanoma cells at high doses, but synchronizes cells at G₁ at non-toxic doses (2.5 or 5 g/ml). We tested the lethality to B16 mouse melanoma cells of combinations of PGD₂ with anticancer drugs. The drugs selected were mostly those used in treating human melanoma: actinomycin D, Bleomycin, BCNU, cis-platin, melphalan, 5-fluorouracil, and 1--D-arabinofuranosylcytosine (ara-C). PGD₂ was combined with the drugs according to 3 different protocols:Protocol 1 An asynchronous culture was given a long term (24 hr) exposure simultaneously to PGD₂ + drug. Combinations with Bleomycin, ara-C or melphalan were additive or slightly antagonistic whereas PGD₂ plus actinomycin D was significantly antagonistic.Protocol 2 Cells synchronized in G₁ by 24 hr PGD₂ exposure were then given a short-term (2 hr) treatment with PGD₂ + drug. Combinations with cis-platin, Bleomycin, BCNU or 5-fluorouracil were additive or slightly antagonistic, whereas melphalan and actinomycin D combinations were significantly antagonistic.Protocol 3 Cells were released from a PGD₂-induced G₁ block and were exposed to drug at different times during cell progression. Actinomycin D was antagonistic when added immediately after release from the G₁ block, but was significantly synergistic when added 10 to 12 hr later.The effect of the combinations cannot be explained by available cell cycle or biochemical information. The antagonism between PGD₂ and several of the drugs resembles the cytoprotective effect of PGD₂ towards various noxious agents.     

Selection of intestinal intraepithelial lymphocyte T cell receptors: evidence for a dynamic tissue-specific process

An extensive comparison of TCRß V-reglon usage byCD8ß^-CD4⁺CD8⁺ intraepithelial lymphocytes (IEL), CD4^-CD8⁺IEL, and lymph node (LN) T cell subsets in three minor lymphocytestimulating (MIs)-disparate, MHC-ldentical mouse strains revealednovel TCR selection patterns. In cases where forbidden V regionswere expressed by CD8ß^- CD4^-CD8⁺ IEL, the same TCRswere deleted from CD8ß^– CD4⁺CD8⁺ IEL, Indicatingthat lack of CD8ß expression was not solely responsiblefor forbidden V-region expression. These results also suggestedthat CD4 may be involved in negative selection of CD4⁺CD8⁺ IELTCRs. In C57BR/cdJ (Mls-1^b2^b) mice, a major increase in V_ß3⁺CD4⁺CD8⁺IEL but not in other IEL or LN subsets was noted suggestinga subset-specific expansion of V_ß3⁺ cells. Negativeselection of V_ß14⁺ cells in only the CD4⁺CD8⁺ IELsubset further supported the existence of intestine-specificTCR selection processes. Analysis of V-reglon expression ofCD8ß⁺ and CD8ß^–CD4^–CD8⁺ IELsubsets revealed that forbidden V-region expression was notstrictly confined to the CD8ß^– subset in allcases. Overall, the data point to a dynamic, gut-specific TCRselection process that may be antigen driven.     

Characterization of B16 melanoma cells resistant to the CC-1065 analogue U-71,184

U-71,184 is a CC-1065 analogue which is highly cytotoxic in vitro and has a broad spectrum of antitumor activity in vivo. Against B16 cells, U-71,184 was 8-fold and 253-fold more potent than Actinomycin D and Adriamycin, respectively. U-71,184 killed 90% of B16 cells at 0.01 ng/ml levels of drug in the medium, which was equivalent to an intracellular concentration of about 8 pg/10(6) cell (= 2 x 10(-8) pmol/cell). A B16 cell line resistant to U-71,184 developed after 3 months of in vitro exposure to gradually increasing concentrations of the drug. The sensitive and resistant cell lines were cloned and a B16/R clone was selected which was 60 to 100 times more resistant to U-71,184 than the cloned sensitive parent (B16/S). Cells grown in the absence of U-71,184 for 2 months retained resistance to the drug. B16/R was slightly cross-resistant only to Adriamycin but not to Actinomycin D, vinblastine, or colchicine. Among alkylating agents, it was slightly cross-resistant to Melphalan but not to 1,3-bis(2-chloroethyl)-1-nitrosourea or cisplatin. B16/R did not overexpress mdr mRNA. Therefore, this cell line does not exhibit the multidrug-resistant phenotype. Most karyotypes of B16/R had a marker chromosome which carried an aberrantly staining region apparently containing repetitive replication of the same segment. Resistance can be partly accounted for by the approximately 10-fold lesser uptake of [3H]-U-71,184 in B16/R, as compared to B16/S. B16/R was cross-resistant in varying degrees to several other CC-1065 analogues. The ratio of the 50% lethal dose of U-71,184 for B16/R, as compared to B16/S, was about 60 (i.e., R/S = 60). In comparison, the following compounds had an R/S ratio of less than 20 (i.e., modest level of cross-resistance to U-71,184): U-68,819, U-73,975, U-75,500, U-75,559, and CC-1065. In contrast, the following compounds had an R/S ratio greater than 20 (i.e., highly cross-resistant to U-71,184): U-71,184 analogues U-71,185, U-73,903, and U-75,012; U-73,975 analogues U-75,613, U-75,032, and U-73,896; and CC-1065 enantiomer U-76,915. We cannot yet explain the difference in the level of cross-resistance between these compounds in vitro. B16/S and B16/R cells were tumorigenic in mice and B16/R was resistant to U-71,184 in vivo. There was no clear indication of cross-resistance of B16/R in vivo to Adriamycin, Actinomycin D, cisplatin, or Melphalan. However, U-73,975, a compound with modest cross-resistance in vitro, was significantly cross-resistant in vivo.     

P388 leukaemia cells resistant to the anthracycline menogaril lack multidrug resistant phenotype

Menogaril is an anthracycline presently in Phase II clinical trials. Menogaril-resistant mouse leukaemia P388 cells were developed in vitro by 4 months of exposure to step-wise increasing concentrations of menogaril after which resistant cells (P388/MEN) were cloned in 320 ng ml-1 menogaril. P388/MEN cells were 40-fold more resistant to menogaril in vitro compared to P388/O and were also resistant in vivo. Resistance to menogaril was stable for at least 2 months in the absence of the drug. The results indicate that P388/MEN, although resistant to an anthracycline, did not display the typical multidrug resistant phenotype. It was not cross-resistant to several structurally unrelated drugs such as actinomycin D, cisplatin, or vinblastine, but it was cross-resistant to the anthracycline, adriamycin. Uptake and efflux of menogaril was similar in sensitive and resistant cell lines. Also, resistance was not reversed by verapamil. No major karyotypic difference was noted between P388/O and P388/MEN. There was no significant amplification or overexpression of the mdr gene in P388/MEN compared to P388/O. In contrast to P388/MEN, P388 cells resistant to adriamycin displayed the typical multidrug resistant phenotype. Glutathione content of P388/MEN cells was similar to that of P388/O and depletion of glutathione did not potentiate menogaril cytotoxicity. Therefore, we conclude that glutathione is not likely to be involved in menogaril resistance to P388/MEN cells.     

Effects of U-71,184 and several other CC-1065 analogues on cell survival and cell cycle of Chinese hamster ovary cells

CC-1065 is a very potent antitumor antibiotic which selectively binds in the minor groove of DNA with alkylation at N-3 of adenine. Since therapeutic doses of CC-1065 caused delayed deaths in mice, analogues were synthesized, some of which had significant antitumor activity. The effects of several of these analogues on inhibition of CHO cell survival, cell progression, and their phase-specific toxicity are reported. CC-1065, U-66,664, U-66,819, U-66,694, and U-71,184 all have a left hand segment with an intact cyclopropyl group but have different tail segments. Lethality of these compounds after 2 h drug exposure was in the following order (50% lethal dose in nM in parentheses): CC-1065 (0.06) greater than U-71,184 (1.3) greater than U-66,694 (3.2) greater than U-68,819 (171) greater than U-66,664 (greater than 1200). In general, these compounds did not inhibit progression from G1 to S but slowed progression through S and blocked cells in G2-M. The phase-specific toxicity of U-71,184 and U-66,694 was different from that of CC-1065. CC-1065 was most cytotoxic to cells in M and early G1 and toxicity decreased as cells entered late G1 and S. In contrast, U-66,694 and U-71,184 were most toxic to cells in late G1. The biochemical and cellular effects of U-71,184 were then studied in detail since it was the most active among these analogues. After a 2-h exposure to 3 ng/ml U-71,184, 90% cell kill or growth inhibition was observed whereas 100 ng/ml was needed for similar inhibition of DNA and RNA synthesis. This discrepancy between the doses suggested that inhibition of nucleic acid synthesis may not be causally related to lethality. Further studies showed that when drug was removed after 2 h exposure, DNA synthesis continued to be inhibited whereas RNA and protein synthesis reached levels higher than the control. Therefore, it is likely that at cytotoxic doses the low level of inhibition of DNA synthesis combined with the stimulation of RNA and protein synthesis leads to unbalanced growth and cell death.     

Understanding How Latino Parents Choose Beverages to Serve to Infants and Toddlers

To determine Latino parents’ beliefs on the health effects of beverages on infants and toddlers, their sources of information on beverages and perceived barriers to following guidelines for healthy beverage consumption by children. We conducted 29 interviews with parents of Latino children ages 6–36 months. Parents were recruited in three community health centers in Northern California. The interviews were recorded, transcribed and analyzed using standard qualitative methods. The following dominant themes emerged. Parents believed that water and milk were healthy beverages for children and that sugar-sweetened beverages (SSBs) were unhealthy. Views on 100 % fruit juice were mixed. Parents distinguished between homemade beverages such as “agua fresca” which they considered healthy, despite containing added sugar, and beverages from stores which were viewed as unhealthy. Participants’ main source of information on beverages was the federal nutrition program for Women, Infants, and Children (WIC). Parents were confused, however, as to why WIC provides juice yet counseled parents to avoid giving their children juice. Parents preferred to receive information on beverages from experts. Differing practices among family members regarding which beverages they provide to children was the most important barrier to following beverage guidelines. Our study suggests that Latino parents are receptive to counseling on beverages from expert sources. Such counseling should address both store-bought and homemade beverages. The WIC program is a key source of information on beverages for Latino parents; thus counseling offered by WIC should be evidence-based and avoid mixed messages.