Hurler's syndrome: a genetic study of clones in cell culture with particular reference to the Lyon hypothesis.

Clones of skin fibroblasts from normal individuals, patients with different mucopolysaccharidoses, and certain of their relatives have been examined for cellular metachromasia and cellular uronic acid. All the clones derived from affected individuals and heterozygous carriers in families with the autosomal forms of Hurler's syndrome showed marked metachromasia and increased cellular uronic acid. Since only one cell population was demonstrated in clones derived from heterozygous carriers, no evidence for autosomal inactivation was obtained. Clones derived from affected individuals with the X-linked recessive form of Hurler's syndrome contained uniform populations of metachromatic staining cells which demonstrated increased cellular uronic acid. Clones derived from the noncarrier fathers showed no cellular metachromasia or increased cellular uronic acid. Clones derived from the heterozygous mothers and sisters showed two populations both qualitatively and quantitatively. On the average, 72% of these clones were metachromatic and demonstrated an increased uronic acid content; 28% of the clones showed no metachromasia and the uronic acid content was similar to that found in normal individuals. The appearance of two distinct cell populations in clones derived from females heterozygous for the X-linked recessive form of Hurler's syndrome provides evidence in favor of the Lyon hypothesis.

Cystic fibrosis of the pancreas. A study in cell culture.

103 individuals from 16 families with cystic fibrosis and 87 individuals without family history of cystic fibrosis have been studied using the methods of cell culture. Skin fibroblast cultures derived from 19 affected children, and fibroblast cultures from 11 different organs obtained at autopsy from two affected children, showed cellular metachromasia. The morphological appearance and the intracellular mucopolysaccharide content enabled these cultures to be divided into two distinct classes. Class I had discrete cytoplasmic metachromatic vesicles and a mucopolysaccharide content similar to that observed in normal fibroblasts. In class II the metachromasia was present in both vesicles and granules and was evenly distributed throughout the cytoplasm. The mucopolysaccharide content of these cells was markedly increased. The cultures derived from the parents, presumed heterozygotes, and other members of each family showed cells with the same type of metachromasia as that demonstrated by the propositus. These data strongly suggest that cystic fibrosis is not a homogeneous entity and, moreover, can be caused by homozygosity of genes at two distinct loci. The recognition of cytoplasmic abnormalities in skin fibroblasts derived from affected individuals and heterozygous carriers for cystic fibrosis should facilitate genetic and biochemical studies on the heterogeneity of this inborn error of metabolism.

Oyster ciliary inhibition by cystic fibrosis culture medium.

Using the oyster ciliary test, sera and fibroblast culture fluid from certain homozygotes and heterozygotes for cystic fibrosis can be shown to contain an inhibitory factor. The tissue culture fluid derived from the metachromatic fibroblast cultures (cystic fibrosis classes I and II) show ciliary inhibition. The fluid derived from the ametachromatic fibroblast cultures (cystic fibrosis class III), thus far studied, cannot be invariably distinguished from the normal noncarriers. Similarly sera from classes I and II show marked ciliary inhibition, whereas sera from class III individuals do not consistently. These experiments support the notion of genetic heterogeneity in cystic fibrosis. The nature of the inhibitory factor in tissue culture fluid and its relationship to the serum factor remain to be explored.

Hurler's syndrome. A genetic study in cell culture.

Seven families affected with Hurler's syndrome have been studied using the methods of cell culture. Skin fibroblasts obtained from the skin of 7 patients with Hurler's syndrome contained metachromatic granules when stained for mucopolysaccharides with toluidine blue O and alcian blue, whereas fibroblasts from normal subjects contained no metachromatic granules. In four families skin cultures of the clinically normal parents showed fibroblasts which contained demonstrable metachromatic granules and "gargoyle" cells and were considered to be heterozygous for the abnormal gene. Fibroblast cultures from certain other members of these families showed metachromasia. These findings were also considered to indicate heterozygosity for the abnormal gene. Three families of the X-linked type of the disease were studied. Fibroblasts cultured from the father contained no metachromatic granules whereas those of the hemizygous mother contained both metachromatic granules and "gargoyle" cells. In one family the abnormal gene could be traced through unaffected individuals for three generations. The prolonged preservation of the biochemical trait in tissue culture will permit studies to be performed designed to clarify the primary action of the abnormal genes which result in Hurler's syndrome, as well as to increase the usefulness of this trait in mapping the human X chromosome.

Hurler's syndrome. Effect of retinol (vitamin A alcohol) on cellular mucopolysaccharides in cultured human skin fibroblasts.

Skin fibroblasts from eight families with Hurler's syndrome (X-linked recessive and autosomal recessive) and normal individuals have been studied in cell culture. A good correlation between cellular metachromasia and the quantitative estimation of intracellular mucopolysaccharides was observed provided the culture conditions were standardized. In both forms of Hurler's syndrome intracellular mucopolysaccharide content could be used to distinguish the affected individual and the carrier from the normal subject, although the total mucopolysaccharide content of fibroblast cultures or the amount in the culture medium did not permit such a distinction. Retinol, in concentrations similar to those encountered in man in hyper-vitaminosis A, caused a reduction in total mucopolysaccharide content of fibroblast cultures from normal and affected individuals. Cultures from three patients with the X-linked recessive form of Hurler's syndrome showed a gradual but marked decrease in cellular metachromasia and approximately 60% decrease in mucopolysaccharide content. Synthesis of polysaccharides and sulfation appeared to be equally affected. On removal of retinol from the medium the content of intracellular mucopolysaccharides returned to pretreatment levels. The possible relevance of these findings to the treatment of Hurler's syndrome is discussed.

Further studies on metachromasia in cultured human fibroblasts. Staining of glycosaminoglycans (mucopolysaccharides) by Alcian blue in salt solutions.

Staining with Alcian blue in various concentrations of magnesium chloride (alcianophilia) has been found to be a useful supplement to metachromatic staining to detect increased cellular concentrations of glycosaminoglycans (mucopolysaccharides). In many instances alcianophilia at 0.3 M MgCl(2) is more specific than metachromasia and does not give "false positives" sometimes found in normal individuals and in those with cystic fibrosis, Gaucher's disease, familial amaurotic idiocy, and pseudoxanthoma elasticum. On the other hand, it gives a "false negative" reaction in the Sanfilippo syndrome (perhaps because the characteristically elevated glycosaminoglycan in this disease, heparan sulfate, is not synthesized by cultured skin fibroblasts), and in the Marfan syndrome. It detects the Maroteaux-Lamy syndrome, which metachromasia does not. The "false positives" given by metachromasia in all six families studied thus far are genuine, reproducible reactions that can be traced through at least three generations of normal individuals within a family. There is therefore, in these families, a genetic factor that causes such metachromasia, but it is not increased glycosaminoglycan concentration.

The Hurler syndrome: a study of cultured lymphoid cell lines.

Lymphoid suspension lines have been established from three patients with the Hurler syndrome and four normals. The Hurler lines can be distinguished from normals by (a) staining characteristics, (b) increase in total cellular mucopolysaccharide content, and (c) increase in dermatan sulfate. Hyaluronic acid is absent in cultured lymphoid cells from normal persons and patients with the Hurler syndrome. The availability of biochemically marked suspension cultures should prove useful for enzymatic studies as well as for further elucidation of this clinical syndrome.

Characterization of cystic fibrosis factor and its interaction with human immunoglobulin.

Cystic fibrosis factor activity (CFFA), assayed as the ability to stop oyster ciliary movement, was present in serum-free medium from actively growing cystic fibrosis skin fibroblast cultures. CFFA was associated with a low molecular weight, negatively charged molecule that contained no uronic acid and was heat and pH labile. When CFFA-positive media were mixed with human IgG1, the CFFA was chromatographically displaced and emerged with the IgG1 fraction on column chromatography. Experiments in which various immunoglobulins were added to CFFA-positive culture media and then incubated with specific anti-immunoglobulins suggested that CFFA binding was class specific for human IgG, subclass specific for IgG1 and IgG2, and occurred with intact unaggregated heavy chains but not with kappa- and lambda-light chains, or Fab, Fc, and F(ab')(2) fragments. The serum protein beta(2)-microglobulin, which has structural homology to IgG, also bound CFFA.

Gaucher's disease: a genetic disease detected in skin fibroblast cultures.

Skin fibroblasts from three adult patients with chronic noncerebral Gaucher's disease, three children of one of the patients, three parents, and six normal individuals were grown in cell culture. Giant fibroblasts containing metachromatic material were seen in all cultures derived from affected individuals and heterozygous carriers but not in those derived from normal individuals.

White blood cell cultures in genetic studies on the human mucopolysaccharidoses.

Cultures of white cells derived from peripheral blood of individuals homozygous and heterozygous for the inherited mucopolysaccharidoses revealed a distinct intracellular metachromatic staining with toluidine blue O. These short-term cultures circumvent the technical problems of skin fibroblast cultures and provide a simple screening procedure to detect the heterozygous state for the mucopolysaccharidoses, as well as offering an opportunity to study the heterozygous state of various inherited storage diseases.

Progeria: a cell culture study on aging.

Progeria is an autosomal recessive disorder showing precocious senility. The cultured skin fibroblast from both the homozygous affected individual and the heterozygous parents can be distinguished from normals by decreased cell growth in culture. Mitotic activity, DNA synthesis, and cloning efficiency are markedly reduced.

Long-term-cultured colon epithelial cell lines from individuals with and without colon cancer genotypes.

In 1982 the characteristics of the first epithelial line established from normal human colon mucosa were reported. The purpose of this report is to describe 5 additional lines established from individuals with and without genotypes associated with genetic predisposition for colon cancer [1 familial polyposis coli (FPC) patient, 1 hereditary nonpolyposis colon cancer (HNPCC) patient, 2 clinically normal individuals (each at risk for one of these autosomal dominant syndromes), and 1 clinically normal individual without any family history of cancer]. The cultured cell lines had the morphologic features associated with epithelium as previously described in the 1982 report. The cell lines derived from genetically (FPC or HNPCC) predisposed individuals had three distinctive characteristics as compared to those derived from individuals without a family history of colon cancer: 1) a higher saturation density (P less than .01); 2) increased in vitro tetraploidy, an in vitro biomarker associated with genetic predisposition for colon cancer; and 3) a higher concentration of carcinoembryonic antigens (CEA) in their conditioned culture medium (P less than .01). Lines derived from normal individuals at risk for the same syndromes were found to have an increased risk status on the first two characteristics. However, the level of CEA did not increase in their culture media, which suggested that the increase in CEA was related to mucosal changes occurring in the preneoplastic sequence in colon cancer.

The Gardner syndrome: a family study in cell culture.

The occurrence of tetraploidy was studied in skin cultures containing both epitheloid and fibroblastic cells derived from 137 members (28 clinically affected, 50 normals at risk and 59 normals not at risk) of 6 families with the Gardner syndrome (3 classical, 3 variant). The cultures from all 28 affected members showed increased tetraploidy. Among the 50 normal members at risk for inheriting the Gardner gene, the cultures from 19 had increased tetraploidy and those from 31 did not. Cultures from 56 of the 59 family members not at risk did not show increased tetraploidy. Although the families were divided into subgroups (classical and variant) on clinical phenotypes, no such subdivision could be made on the basis of increased tetraploidy in skin cultures.

Epithelial line from normal human colon mucosa.

An epithelial line was established from an explant culture derived from human colon mucosa exhibiting a normal phenotype by taking advantage of two differences in in vitro cell behavior of mixed cell (epithelial-fibroblast) monolayer [rate of adherence to a plastic surface from a suspension and toxicity to collagenase (25 U/ml medium) in the culture medium]. Cells with an epithelial morphology could be identified at all times throughout the first 5 culture months--first as epithelial sheets migrating from explants, then as small clusters within the fibroblast monolayer, and finally after 5 months of selective culturing as the only cell type present. The line maintained an epithelium-like morphology and growth pattern through 27 continuous culture months (35 subcultures) and was clearly distinguishable from colon fibroblast cultures grown under the same conditions. The ultrastructural features of this line that grew as a continuum included extensive membrane interdigitations with intercellular junctions, tonofilaments, mucus droplets, no Weibel-Palade bodies (characteristic of endothelial cells), and microridges uniformly covering the cell surfaces; all of these features were absent in colon fibroblast cultures. Growth kinetics (generation time, saturation density, and life-span) were distinctly different from those of colon fibroblast cultures grown under the same conditions. During 27 continuous culture months, the cells remained diploid (2-3% hyperdiploidy) and showed no evidence of senescence.

Tetraploidy in cultured dermal fibroblasts from patients with heritable colon cancer.

The incidence of tetraploidy in monolayer cultures of dermal fibroblasts from 40 clinically affected members from 10 families with heritable colon cancer was compared with similar cultures from 40 clinically normal volunteers with three different techniques: (1) metaphase assay (MA), (2) flow cytometry of stationary cell cultures (FCMs), and (3) flow cytometry of proliferating cell cultures (FCMd). In vitro tetraploidy was considered to be present (IVT+): (1) by MA if more than 7 per cent of metaphases were tetraploids, (2) by FCMs if more than 8 per cent of cells in stationary cultures were tetraploids (i.e. DNA index greater than 1), and (3) by FCMd of more than 8 per cent of cells in logarithmic cultures were tetraploids (i.e. DNA index greater than 2). There was excellent concordance between the three assays, which assigned all the 40 HCC patients to the IVT+ category and all the 40 normal individuals to IVT- category. This in vitro data on the incidence of IVT in clinically affected members from HCC families suggested that this putative biomarker for colon cancer proneness may ultimately be useful in identification of such increased genetic risk for colon cancer in such HCC families and further supported the hypothesis that germinal mutations for cancer proneness (detected by in vitro expression of IVT) are relevant in the development of HCC.

A standardized assay to identify colon cancer genotypes by in vitro tetraploidy in human dermal fibroblasts.

Increased tetraploidy in dermal fibroblast cultures (IVT), which is a putative biomarker for genetic predisposition to colon cancer, was assayed by two different methods: by per cent of tetraploid metaphases in chromosome preparations from non-confluent monolayer petri dish cultures in logarithmic growth (MA); by flow cytometry (FCM) distribution of DNA content of propidium iodide stained cells from plastic flask cultures in stationary growth phase. The assayed cultures were derived from split thickness skin biopsies from two clinical groups: 11 normal individuals without a family history of colon cancer and 17 clinically affected individuals with autosomal dominant polyposis coli cancer syndrome (6 with colonic adenomatosis, i.e. familial polyposis coli (FPC) and 11 also having extracolonic lesions, i.e. the Gardner syndrome, (GS). Concordance was observed between the two assays. There was a linear relationship between the per cent of tetraploid metaphases assayed in the chromosome preparations and the per cent of cells with a DNA index (DI) of precisely 2, or of all cells with a DI greater than 1 as determined by FCM. In comparisons between the clinical groups, there was significant differences in the FCM DNA index (p values less than 0.001) of IVT- (normals and IVT- FPC) and IVT+ individuals (IVT+ GS and those FPC with IVT). The per cent of cells with a DI of 2 and greater than 1 could be used to distinguish all IVT- individuals (normals and IVT- FPC) from IVT+ FPC individuals. However, only by per cent of cells with a DI greater than 1 could all the individual GS affecteds be distinguished from IVT- individuals. Thus, the per cent of cells with a DI greater than 1 is considered to be the parameter of choice to be used in assaying IVT by flow cytometry.

In vitro evidence of heterogeneity within pancreatic cancer.

The relative importance of the genome and environmental factors has not been established in pancreatic carcinogenesis. The identification of in vitro expressions of cancer genes should enhance such clarification. Hyperdiploidy, with the exception of a low incidence (0%-7%) of tetraploidy, has rarely been observed in dermal monolayer cultures from normal individuals without a family history of solid tumors. Hyperdiploidy (incidence over 1% in a culture) was not observed in dermal fibroblast monolayer cultures derived from 143 clinical normals (ages 18-80 years). In vitro hyperdiploidy (7%-14%), with a normal occurrence of tetraploidy (0%-3%), was observed in 6 of 28 patients with acinar adenocarcinoma of the body of the pancreas. No in vitro hyperdiploidy was observed in the remaining pancreatic cancer patients or in the 25 spouses with negative family cancer histories studied. As hyperdiploidy with a normal occurrence of tetraploidy has been previously reported [1] in a familial pancreatic cancer cluster, this chromosomal alteration was considered to be an in vitro expression of genetic predisposition for pancreatic cancer in approximately 20% of this small (28 patients) sample. In vitro studies on a significantly larger patient sample will be required to determine its incidence in a patient group with acinar adenocarcinoma of the body of the pancreas.

In vitro hyperdiploidy in a family with nasopharyngeal cancer.

In this in vitro study of 28 members of a family with nasopharyngeal cancer (NPC) and nasopharyngeal angiofibromas (NPA) in consecutive generations, in vitro hyperdiploidy, (IVH) in dermal fibroblast cultures was associated with NPC in 2 affected members, NPA in 4 affected members, and 4 of 16 members considered to be at increased genetic risk for these tumors. None of the six controls, all spouses, had IVH. As IVH has been shown to be associated with high specificity (approximately 99%) with other heritable single tumors, IVH appeared to be a potential biomarker also for genetic predisposition for NPC. As NPC and NPA did not occur in a single affected family member, any association other than with IVH has not been established. IVH may be the in vitro expression of a gene/genes conveying tumor proneness per se, or it may determine tissue specificity of tumor pathology. As cellular hyperdiploidy has been considered to convey genomic instability, chromosome instability in numerically normal (diploid) and altered (tetraploidy and hyperdiploid) dermal fibroblasts from two NPC-affected members and two control spouses were evaluated by a parameter of chromosome stability--the frequency of sister chromatid exchanges (SCE) per cell. Irrespective of donor source, the diploid and tetraploid fibroblasts exhibited little evidence of chromosome instability, whereas there was a statistically significant difference in the mean frequency of SCE/cell between hyperdiploid and diploid cells in cultures derived from NPC-affected members (p less than 0.01).

Increased in vitro tetraploidy in dermal monolayer cultures derived from normals.

Increased in vitro tetraploidy has been considered an in vitro expression of some cancer-prone genes in dermal monolayer cultures. (Danes BS, Alm T, Scand J Gastroenterol 16, 421-427, 1981; Danes BS, Cancer 48, 1596-1601, 1981). Its occurrence was determined in cultures established from skin biopsies from 112 normals (university students and personnel) whose family cancer histories were known to ascertain its relevance to cancer occurrence based on pedigree data. Of the 40 who gave no family cancer history, one showed increased in vitro tetraploidy (1% of the total studied; 2.5% of those without a family cancer history). Of the 72 with one or more first- or second-degree relatives with a family cancer history, 14 showed this alteration (12.5% of the total studied; 19% of those with a family cancer history). Increased in vitro tetraploidy was found in some normals from families with lung, breast, genital system, and gastrointestinal tract cancers. It may prove ultimately to be relevant to include such in vitro information in medical surveillance and cancer management programs.

Evidence for genetic predisposition for some nasopharyngeal cancers by in vitro hyperdiploidy in human dermal fibroblasts.

A numerical alteration in chromosome complement in human dermal fibroblast cultures, hyperdiploidy with a normal occurrence of tetraploidy (IVH), has been reported to be associated with some hereditary single tumors including squamous carcinoma of the nasopharynx (NPC). Its incidence was compared in cultures derived from 39 NPC patients and 29 clinically normal subjects without a family cancer history by two different methods (percentage of numerically altered metaphases in chromosome preparations from nonconfluent monolayer Petri dish cultures in logarithmic growth, and by the distribution of DNA content of propidium iodide stained cells from plastic flask cultures in stationary growth phase as assayed by flow cytometry) to ascertain its usefulness in identification of such genetic predisposition. Concordance was observed between the two assays. There was a linear relationship between the percentage of hyperdiploid metaphases assayed in the chromosome preparations and the percentage of cells with a DNA index of greater than 1 as determined by flow cytometry. By metaphase assay, none of the 29 normals showed IVH. OF the 39 carcinoma of the nasopharynx patients studied, 19 had IVH and 20 did not. By flow cytometry there were significant differences in the flow cytometry DNA index (p less than 0.001) of IVH-negative (all normals and 20 carcinoma of the nasopharynx patients) and IVH-positive carcinoma of the nasopharynx patients. The percentage of cells with a DNA index of 2 and greater than 1 could be used to distinguish all IVH-negative from the IVH-positive subjects and, thus, were considered to be the parameters of choice in assaying IVH by flow cytometry. None of the subjects studied showed increased in vitro tetraploidy (IVT), which has been associated with some heritable colon cancer syndromes. Irrespective of family cancer history, approximately one-half of the NPC patients (19 of 39) had IVH, which has been reported to be associated with the in vivo expression of certain heritable tumors including carcinoma of the nasopharynx. The average age of carcinoma of the nasopharynx diagnosis was earlier (mean 52 yr) for the IVH-positive group than for the IVH-negative carcinoma of the nasopharynx group (mean 67 yr).