For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

https://cancerres.aacrjournals.org/content/canres/48/3/589.full.pdf

Feasibility of Drug Screening with Panels of Human Tumor Cell Lines Using a Microculture Tetrazolium Assay

We have reconstituted a matrix of basement membrane onto a filter in a Boyden chamber and assessed the ability of various malignant and nonmalignant cells to penetrate through the coated filter. Cells from all the malignant cell lines tested were able to cross the matrix in 5-6 h, whereas human fibroblasts as well as mouse 3T3 and 10T1/2 cell lines, which are not tumorigenic, were not invasive. In addition, normal primary prostate epithelial cells and benign prostatic hyperplasia cells were not invasive when tested in this assay, whereas malignant prostate carcinoma cells were highly invasive. Parallel experiments with these prostatic cells using the intrasplenic assay for metastasis detection in the nude mouse confirmed the benign behavior of the former cells and the metastatic phenotype of the latter ones. These results suggest that this in vitro test allows the rapid and quantitative assessment of invasiveness and a means to screen for drugs which alter the invasive phenotype of tumor cells.

https://cancerres.aacrjournals.org/content/canres/47/12/3239.full.pdf

A Rapid in Vitro Assay for Quantitating the Invasive Potential of Tumor Cells

The LNCaP cell line was established from a metastatic lesion of human prostatic adenocarcinoma. The LNCaP cells grow readily in vitro (up to 8 x 10(5) cells/sq cm; doubling time, 60 hr), form clones in semisolid media, are highly resistant to human fibroblast interferon, and show an aneuploid (modal number, 76 to 91) human male karyotype with several marker chromosomes. The malignant properties of LNCaP cells are maintained. Athymic nude mice develop tumors at the injection site (volume-doubling time, 86 hr). Functional differentiation is preserved; both cultures and tumor produce acid phosphatase. High-affinity specific androgen receptors are present in the cytosol and nuclear fractions of cells in culture and in tumors. Estrogen receptors are demonstrable in the cytosol. The model is hormonally responsive. In vitro, 5 alpha-dihydrotestosterone modulates cell growth and stimulates acid phosphatase production. In vivo, the frequency of tumor development and the mean time of tumor appearance are significantly different for either sex. Male mice develop tumors earlier and at a greater frequency than do females. Hormonal manipulations show that, regardless of sex, the frequency of tumor development correlates with serum androgen levels. The rate of the tumor growth, however, is independent of the gender of hormonal status of the host.

https://cancerres.aacrjournals.org/content/canres/43/4/1809.full.pdf

LNCaP model of human prostatic carcinoma.

During 1985-1990 the U.S. National Cancer Institute (NCI) phased out its murine leukemia P388 anticancer drug screening program and developed as the replacement a new in vitro primary screen based upon a diverse panel of human tumor cell lines. For each substance tested, the screen generates a remarkably reproducible and characteristic profile of differential in vitro cellular sensitivity, or lack thereof, across the 60 different cell lines comprising the panel. Several investigational approaches to display, analysis and interpretation of such profiles and databases, derived from the testing of tens of thousands of substances during the past 4-5 years since the NCI screen became fully operational, have been explored. A variety of useful, practical applications of the in vitro screen have become apparent. As these applications continue to evolve, they are proving to be complementary to diverse other anticancer screening and drug discovery strategies being developed or pursued elsewhere. Reviewed herein are some practical considerations and selected specific examples, particularly illustrating research applications of the NCI screen that may be more broadly applicable to the search for new anticancer drug development leads with novel profiles of antitumor activity and/or mechanisms of action.

Some Practical Considerations and Applications of the National Cancer Institute In Vitro Anticancer Drug Discovery Screen

The tumor suppressor gene CDKN2/p16/MTS1, located on chromosome 9p21, is frequently inactivated in many human cancers through homozygous deletion. Recently, we have reported another pathway of inactivation that involves loss of transcription associated with de novo methylation of a 5' CpG island of CDKN2/p16 in lung cancers, gliomas, and head and neck squamous cell carcinomas. We now show that this aberrant CpG island methylation also occurs frequently in cell lines of breast cancer (33%), prostate cancer (60%), renal cancer (23%), and colon cancer (92%) and is associated with loss of transcription. Primary tumors of the breast (31%) and colon (40%) also displayed de novo methylation of this CpG island. This alteration of p16 in colon cancer was particularly striking, since inactivation does not occur through homozygous deletion in this tumor type. Our data show that in tumors, de novo methylation of the 5' CpG island is a frequent mode of inactivation of CDKN2/p16 and also firmly demonstrate that CDKN2/p16 is one of the most frequently altered genes in human neoplasia.

https://cancerres.aacrjournals.org/content/canres/55/20/4525.full.pdf

Inactivation of the CDKN2/p16/MTS1 Gene Is Frequently Associated with Aberrant DNA Methylation in All Common Human Cancers

A long-term tissue culture cell line has been derived from a human prostate adenocarcinoma metastatic to the brain. The cell line, DU 145, has been passaged 90 times in vitro over a period of 2 years. The cells are epithelial, grow in isolated islands on plastic Petri dishes, and form colonies in soft agar suspension culture. Karyotypic analysis demonstrates an aneuploid human karyotype with a modal chromosome number of 64. Distinctive marker chromosomes (a translocation Y chromosome, metacentric minute chromosomes and three large acrocentic chromosomes) have been identified. Electron microscopy of the original tumor tissue and of the tissue culture cell line show a remarkable similarity in cell organelle structure.

Isolation of a human prostate carcinoma cell line (DU 145).

Nude mice of NIH/Swiss background were utilized for the heterotransplantation of a tissue culture cell line derived from a human prostate adenocarcinoma metastatic to the brain These cells, which had been grown in vitro for 13 passages, formed solid tumors when injected sc into nude mice The cell line DU 145 has been passaged 60 times in vitro over a period of 18 months Tumors removed from the mice were serially transplanted to additional mice and reestablished in vitro Light-microscopic analysis of the tumor grown in nude mice revealed a strong similarity to the patient's metastatic tumor The ultrastructure of the tumor cells propagated in nude mice was compared to that of the original human tumor cells and to the tissue culture cells, both before and after passage in nude mouse No major differences were detected Karyotypic analysis of the tumor cells grown in vitro before mouse passage, grown in nude mouse, and grown in vitro after mouse passage indicated chromosomal identity and consistent marker chromosomes: three large acrocentric chromosomes and metacentric minute chromosomes

https://cancerres.aacrjournals.org/content/canres/37/11/4049.full.pdf

Heterotransplantation of a Human Prostatic Adenocarcinoma Cell Line in Nude Mice

Characterization of a human prostate adenocarcinoma cell line (DU 145) as a monolayer culture and as a solid tumor in athymic mice.

Through the process of sequential adsorption analysis of antimyelin I antibodies, raised in rabbits, rat myelin I was shown to possess a class of determinants distinct from synaptosomes and brain mitochondria, a class shared with synaptosomes but not brain mitochondria, and a class shared with synaptosomes and brain mitochondria as well as with liver mitochondria. Myelin and synaptosomes were thus distinguished immunologically from mitochondria and from each other. Although most of the antimyelin I antibodies also cross-reacted with myelin II some remained distinctive for myelin I, thus revealing a class of determinants in I not prominent in II. Crossreactions of the rabbit antirat-myelin antibodies with myelin I from different species in increasing order were bullfrog, 23%; chicken, 37%; rabbit, 50%; guinea pig, 50%; dog, 53%; human, 59%; neonatal rat, 61%; mouse, 72%; and hamster, 73%. The last three values were felt to be underestimated.

Don D. Mickey

Papers

Isolation of a human prostate carcinoma cell line (DU 145).

Heterotransplantation of a Human Prostatic Adenocarcinoma Cell Line in Nude Mice

Characterization of a human prostate adenocarcinoma cell line (DU 145) as a monolayer culture and as a solid tumor in athymic mice.

The Specificity and Cross-Reactivity of Antisynaptosome Antibodies as Determined by Sequential Adsorption Analysis

Zonal centrifuge profiles of rat brain homogenates: Instability in sucrose, stability in iso-osmotic Ficoll-sucrose☆