Growth abnormalities of cultured human skin fibroblasts derived from individuals with hereditary adenomatosis of the colon and rectum

Abstract

A heritable propensity to develop malignant lesions is found in individuals with familial adenomatosis of the colon an rectum (ACR) and the Gardner's syndrome variant, an autosomal dominant trait. In the present study, the growth characteristics of cultured skin fibroblasts (SF) derived from normal-appearing flat skin biopsies of ACR families, representing all phenotypes, and appropriate controls were investigated. SF were obtained from stocks between the second and fifth passages and growth to confluency in Eagle's Minimal Essential Medium (EMEM) supplemented with 15% fetal calf serum (FCS). Following trypsinization, cells were replanted in EMEM supplemented with either 1% or 15% FCS at an initial density of 4 x 10(3) cells/cm2 and counted daily for five days. Normal SF representing several age groups (both sexes) and those obtained from non-afflicted individuals of ACR families grew only in 15% FCS. In contrast, SF from ACR subjects and from embryonal skin grew both in 1% and 15% FCS. SF from several clinically asymptomatic adults, children or ACR patients, grew in 1% FCS as well. Cell cultures from ACR individuals showed regions of criss-crossed arrays and multilayered pattern. These growth properties were not observed in normal cell cultures. The SF from ACR individuals did not grow in methocel, nor did they form tumors in athymic mice. These results suggest the occurrence of previously undetected biochemical alterations in SF taken from ACR genotypes.

Saturation Density of Skin Fibroblasts as a Quantitative Screen for Human Cancer Susceptibility

“…SF obtained from symptomatic individuals from families bearing this mutation exhibit growth behavior that distinguishes them from SF of normal individuals in two characteristics, namely an ability to proliferate in low concentrations of fetal bovine serum (FBS) (ref. 7), and a capacity to multiply to significantly higher than normal SD in conventional concentrations of FBS (8). The asymptomatic progeny of symptomatic members of ACR/FAP families would be expected to be, and are, mixed among those that can and cannot proliferate in low FBS (Table 1A), because these progeny consist of carriers and noncarriers of mutations in one allele for the APC gene.…”

“…Procedure for optimizing SD of SF as a quantitative screen for cancer susceptibility. Cultures of SF are established from subepidermal biopsies of human skin by conventional methods (7,9). The SF are divided in two (or four) parallel lineages, 5 × 10 3 cells/cm 2 , in 10% or 15% serum for 2 or 3 wk; and in 2% or 5% serum for 2 wk with frequent medium changes.…”

“…Living cells, however, integrate the phenotypic effects of many genes, and long-standing evidence is presented that increased saturation density (SD, the maximum number of cells in a culture, limited by contact inhibition and the concentration of serum in the medium) of skin fibroblasts (SF) is displayed both in the case of the high penetrance of a single mutated gene in cancerprone families (7,8), and of multiple mutated genes in "sporadic" breast (9) and other cancers (10). Increases in SD result from selection of cells under contact inhibition of mutants that mainly arise during the replication of cells (11,12).…”

Defective organization of actin in cultured skin fibroblasts from patients with inherited adenocarcinoma

“…SF from ACR patients and from about one-half of their children have lost serum-and density-sensitive growth control in culture (6)(7)(8)(9). Compared with SF from normal persons, ACR SF grow better in low serum concentration and grow to a higher density (6).…”

Patients with tuberous sclerosis have fibroblasts with normal limits for growth characteristics and sensitivities to DNA alkylating agents

“…Enhanced growth, i.e. ability to proliferate under suboptimal nutrient conditions, has been recognized in cases of hereditary adenomatosis of colon and rectum (ACR) (Pfeffer et aL, 1976) and retinoblastoma (Kossakowska et al, 1982), which are autosomal dominant disorders. The cells from patients with hereditary retinoblastoma or ACR are possibly hypersensitive to ionizing radiation or DNA alkylating agents (Weichselbaum et al, 1978;Barfknecht and Little, 1982).…”