Use of young nude mice for selection of subpopulations of cells with increased metastatic potential from nonsyngeneic neoplasms.

The purpose of these studies was to investigate whether young (3-wk-old) nude mice, which lack functional T-lymphocytes and demonstrate low natural killer cell activity, could serve as in vivo models for the selection of metastatic subpopulations of cells from heterogeneous allogeneic melanomas. Three-week-old BALB/cAnN or N:NIH(S) nude mice received iv injections of single cells harvested from either the B16 or K-1735 melanoma. Individual pulmonary metastases were harvested 4 weeks later and implanted sc into nude mice to expand the population. Cells from each of these metastases colonized in the lungs of 3-week-old nude mice and 6-week-old normal syngeneic mice with significantly higher efficiency than did cells from the parent tumors. It was concluded that, in addition to being a useful in vivo model for ascertaining the metastatic potential of neoplasms, the healthy young nude mouse could be used for selecting and maintaining tumor cell variants of high metastatic potential room heterogeneous allogeneic tumors.

Production of human chorionic gonadotropin and its subunits by human tumors growing in nude mice.

The production and secretion of human chorionic gonadotropin (HCG) and its subunits by human tumors growing in nude mice have been examined. JAR choriocarcinoma cells growing in nude mice produce both free alpha subunit and complete HCG, but there is a decrease in the amount of free alpha subunit relative to complete HCG produced in vivo compared to HCG subunit production by these cells growing in culture. Cell lines that produce only free alpha subunit in culture (HeLa cervical carcinoma, ChaGo bronchogenic carcinoma, and BT-20 breast carcinoma) continue to produce primarily free alpha subunit in vivo, but a small amount of HCG-beta/HCG is detectable in the 24-hr urine collected from mice bearing HeLa or ChaGo tumors. CBT cells derived from a glioblastoma multiforme produce both alpha and HCG-beta/HCG in vivo. This represents a distinct shift from the pattern of HCG subunit production by CBT cells in culture because cultured CBT cells produce only free beta subunit and do not synthesize either free alpha or complete HCG. Thus, for human tumors growing in nude mice, there appears to be a shift toward more complete HCG production and a decrease in free subunit production as compared to the pattern observed for cultured cells.

Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase.

The epidermal growth factor receptor (EGFR) is overexpressed in a significant percentage of carcinomas and contributes to the malignant phenotype. CP-358,774 is a directly acting inhibitor of human EGFR tyrosine kinase with an IC50 of 2 nM and reduces EGFR autophosphorylation in intact tumor cells with an IC50 of 20 nM. This inhibition is selective for EGFR tyrosine kinase relative to other tyrosine kinases we have examined, both in assays of isolated kinases and whole cells. At doses of 100 mg/kg, CP-358,774 completely prevents EGF-induced autophosphorylation of EGFR in human HN5 tumors growing as xenografts in athymic mice and of the hepatic EGFR of the treated mice. CP-358,774 inhibits the proliferation of DiFi human colon tumor cells at submicromolar concentrations in cell culture and blocks cell cycle progression at the G1 phase. This inhibitor produces a marked accumulation of retinoblastoma protein in its underphosphorylated form and accumulation of p27KIP1 in DiFi cells, which may contribute to the cell cycle block. Inhibition of the EGFR also triggers apoptosis in these cells as determined by formation of DNA fragments and other criteria. These results indicate that CP-358,774 has potential for the treatment of tumors that are dependent on the EGFR pathway for proliferation or survival.

Separation of guinea pig peripheral blood lymphocytes by discontinuous density gradient centrifugation using Ficoll-metrizoate.

We present a simple method for the recovery of lymphocytes from guinea pig peripheral blood by discontinuous density gradient centrifugation using Ficoll-metrizoate solutions. This technique is formulated specifically for the routine preparation of guinea pig lymphocytes for in vitro cultivation and is capable of recovering 40-60% of available lymphocytes. Whole, heparinized (10-100 U/ml) blood was drawn from strain 2 guinea pigs by cardiac puncture, diluted 1 : 4 with Ca2,Mga free Hanks' balanced salt solution (CMF-HBSS) containing EDTA (5 mM) and gentamicin (50 microgram/ml), and incubated at room temperature for 30-60 min to promote leukocyte disaggregation. Five volumes of diluted blood were layered onto 2 vol of Ficoll-metrizoate adjusted to a density of 1.107 g/ml with sodium metrizoate solution. A band of mononuclear cells (80-90% lymphocytes, 10-20% monocytes, and less than 2% granulocytes) formed at the interface after centrifugation (400 X g, 20-40 min, room temperature). More than 95% of the cells were viable by trypan blue exclusion. Lymphocytes recovered from as little as 3-5 ml whole blood were more sensitive to antigen- or mitogen-activated transformation than leukocyte suspensions obtained by dextran-citrate sedimentation with or without nylon column filtration.

N-(5-fluorobenzothiazol-2-yl)-2-guanidinothiazole-4-carboxamide. A novel, systemically active antitumor agent effective against 3LL Lewis lung carcinoma.

N-(5-Fluorobenzothiazol-2-yl)-2-guanidinothiazole-4-carboxam ide (1) is a member of a series of amides found to substantially increase lifespan in mice bearing established micrometastatic 3LL Lewis lung carcinoma. Amide 1 is effective after either oral or intraperitoneal dosing in acute, subacute, or chronic regimens. 1 is well tolerated in this model with an excellent therapeutic index relative to the cytotoxic anticancer drug adriamycin.

Growth responses of Escherichia coli to the surfactant dodecyl benzene sulfonate.

When strains of Escherichia coli are grown in broth cultures containing the anionic surfactant sodium dodecyl benzene sulfonate (NaDDBS), they exhibit unique growth responses. After 20 to 24 hr of incubation, they become slimy and viscous, and an addition of ethanol to the supernatant liquid yields a distinctive white, fibrous precipitate. The production of this material was shown to be dependent on the presence of NaDDBS in the medium. This precipitate from E. coli ATCC 11303 was found to contain 41 to 53% protein, 10 to 11% deoxyribonucleic acid, 6.8 to 7.4% ribonucleic acid, 15 to 25% carbohydrate, and 9% lipid. It is distinctive from naturally occurring E. coli slimes in several respects. Our data suggest that its formation is the primary result of the leakage of intracellular components into the medium. However, the rate of cell proliferation indicates a partial but not complete or lethal lysis. A limited utilization of NaDDBS as a carbon source was also shown.

Studies of the paw test and tumor-associated immunosuppression in mice.

Specific paw test reactivity to tumor cells was transferred to normal mice using lymphoid cells or serum from mice with a growing or surgically excised methylcholanthrene-induced transplantable sarcoma. When the donors had been sensitized to PPD and to tumor, normal recipients of lymphoid cells became reactive by specific paw test to tumor cells and PPD. Normal recipients of serum from the same donors also became reactive to tumor cells. Normal recipients of serum from tumor-bearing mice developed depressed PHA paw tests. Adoptive transfer with cells or serum of paw test reactivity to tumor and PPD was unsuccessful when recipients had large tumors. This was the case when the recipients had either tumor of the same line or that of a separate line that did not cross-react antigenically.

Active specific immunotherapy of established micrometastases with BCG plus tumor cell vaccines: effective treatment of BCG side effects with isoniazid.

Intradermal vaccinations with Bacillus Calmette-Guérin (BCG) and viable but nontumorigenic syngeneic hepatocarcinoma cell vaccines were used successfully to treat micrometastatic disease experimentally induced in inbred guinea pigs at 100 times the minimal lethal dose. Several complications have been associated with the use of viable BCG organisms in the treatment of cancer patients and, in this animal model, intradermal administration of BCG uniformly results in severe ulceration and eschar formation at the injection sites leading to secondary microbial invasion and regional lymphadenopathy. We now report the use of isoniazid (Nydrazid) as part of an active specific immunization regimen. Incorporation of isoniazid into the immunization procedure for two weeks alleviates or reduces the side effects of BCG infection. Moreover, with proper consideration of BCG dosage, isoniazid does not impair the efficacy of the BCG plus tumor cell vaccines.

Inhibition of epidermal growth factor receptor-associated tyrosine phosphorylation in human carcinomas with CP-358,774: dynamics of receptor inhibition in situ and antitumor effects in athymic mice.

Phosphorylation of tyrosine residues on the epidermal growth factor (EGF) receptor (EGFr) is an important early event in signal transduction, leading to cell replication for major human carcinomas. CP-358,774 is a potent and selective inhibitor of the EGFr tyrosine kinase and produces selective inhibition of EGF-mediated tumor cell mitogenesis. To assess the pharmacodynamic aspects of EGFr inhibition, we devised an ex vivo enzyme-linked immunosorbent assay for quantification of EGFr-specific tyrosine phosphorylation in human tumor tissue specimens obtained from xenografts growing s.c. in athymic mice. When coupled with pharmacokinetic analyses, this measurement can be used to describe the extent and duration of kinase inhibition in vivo. CP-358,774 is an effective, orally active inhibitor of EGFr-specific tyrosine phosphorylation (ED(50) = 10 mg/kg, single dose). It has a significant duration of action, producing, on average, a 70% reduction in EGFr-associated phosphotyrosine over a 24-h period after a single 100 mg/kg dose. Inhibition of EGFr phosphotyrosine in an ex vivo assay format effectively estimates the potency and degree of inhibition of EGFr-dependent human LICR-LON-HN5 head and neck carcinoma tumor growth. Substantial growth inhibition of human tumor xenografts was achieved with p.o. doses of the compound (ED(50) = 10 mg/kg q.d. for 20 days). Combination chemotherapy with cisplatin produced a significant response above that of cisplatin alone with no detectable effects on body weight or lethal toxicity. Taken together, these observations suggest that CP-358,774 may be useful for the treatment of EGFr-driven human carcinomas.