Comparison of adhesive bond strength of different cell types in vitro

Summary An established in vitro assay for quantitating cell-substratum adhesion has been utilized to measure the adhesiveness of 10 cell lines to a colloidal overlay. The procedure, a derivation of the William’s blister test for adhesives, involves growing cells to confluency on a polystyrene surface and then overlaying the monolayers with a Bacto-agar substratum. The cell-agar substratum systems are debonded and the^ra,adhesive bond strength, of the separation of the two interfaces calculated. The^ra’s were determined for the following cell types: SGL (gingival epithelial-like), L (transformed mouse fibroblasts), HeLa (human carcinoma), MDCK (canine kidney epithelial), WI-38 (human embryonic lung), Flow 1000 (human embryonic skin—muscle), Flow 4000 (human embryonic kidney), Flow 5000 (whole human embryo), BALB/c 3T3 (mouse fibroblasts) and SV₄₀-transformed BALB/c 3T3 (simian virus 40-transformed mouse fibroblasts). Transformed cells (L, HeLa and SV₄₀-transformed BALB/c 3T3) proved to be quantitatively less adhesive (^ra/cell) than either fibroblast or epithelial-like cell lines. Of the “normal” cells tested the kidney cells, human embryonic fibroblast and canine epithelial cells, and the gingival epithelial-like cells demonstrated a weaker binding to the colloidal overlay than the mouse fibroblasts (BALB/c 3T3), the human embryonic lung, the human embryonic skin-muscle, and the whole human embryo fibroblast cell lines. Concanavalin A increased the bonding strength of Flow 5000 cells after 24 hr incubation; however, the adhesiveness of the treated BALB/c 3T3 cells remained similar to the unterested samples while the^ra of the treated SV₄₀-transformed BALB/c 3T3 cells decreased. This research was supported by National Institute of Dental Research Grant DE03983.     

Flow cytometry analysis of early DNA content changes in human and monkey cells following infection with simian virus 40

Simian virus 40 (SV₄₀) is capable of inducing cellular DNA synthesis in permissive and nonpermissive cells. Utilizing flow cytometry, we analyzed the DNA content changes in two diploid human cell strains and two monkey cell lines. The osteogenesis imperfects (OI) human skin fibroblasts were induced into DNA synthesis, and within one to two cell generations, a polyploid cell population was produced. With WI-38 phase II cells, a similar pattern of increased cycling of cells into DNA synthesis was observed; however, the majority (～60%) of the cells were blocked in the G₂ + M phase of the cell cycle. At later time intervals, an increase in the G₁ population was demonstrated. The two monkey cell lines responded to SV₄₀ virus with an accumulation of cells in the G₂ + M phase of the cell cycle. Thus, two diploid human cell strains exhibited different cell cycle kinetics early after infection with SV₄₀ virus. The one strain (WI-38) behaved similarly to the two monkey cell lines studied. The other strain (OI) responded similarly to nonpermissive (transformin) cells infected with SV₄₀ virus.     

Induction of DNA polymerase α in senescent cultures of normal and Werner's syndrome cultured skin fibroblasts

DNA polymerase α activity was determined following serum stimulation of early and late passages of human diploid fibroblast-like (HDFL) cultures derived from apparently normal donors (two strains) and from a patient with Werner's syndrome (one strain). Induction of this enzyme was observed in both low passage, actively proliferating cultures and in postmitotic “senescent” cultures from all three strains. The maximal polymerase activity of early and late passage cells of each strain were nearly identical when normalized to the number of cells present. However, the activity of the enzyme was observed to be significantly lower in late passage cultures when normalized to total protein content apparently because of enlargement of the senescent cells. The behavior of Werner derived cells was similar to that of the normal cells. The induction of DNA polymerase α in senescent cultures indicates that they retain the capacity to carry out some complex metabolic responses to mitogen stimulation. In addition, these results suggest the possibility that dilution of DNA polymerase α and/or other DNA replication factors may play a role in the onset or maintenance of the postmitotic state in the enlarged senescent HDFL cells.     

Differences in electron transport potential,antioxidant defenses,and oxidant generation in young and senescent fetal lung fibroblasts (WI-38)

The activities and mRNA abundances of enzymes that regulate the rate of electron flow through the electron transport chain (ETC), including NADH dehydrogenase, succinate dehydrogenase, and cytochrome c oxidase, were examined in young and senescent fetal lung fibroblasts (WI-38). We also determined the activities and mRNA abundances of antioxidant defenses including superoxide dismutase, catalase, and glutathione peroxidase. We confirmed our previous report of a senescence-related increase in the abundance of ND4, a mitochondrially encoded subunit of NADH dehydrogenase. The activities of cytochrome c oxidase and NADH dehydrogenase were also elevated in senescent cultures. No differences were observed in the mRNA abundances of COX-1, a mitochondrially encoded subunit of cytochrome c oxidase or of nuclearly encoded subunits of various electron transport components (SD, COX-4, and ND 51). Lucigenin-detected chemiluminescence and H₂O₂ generation were both elevated in senescent cells. Catalase activity was also elevated in senescent fibroblasts. However, no differences in catalase mRNA abundance were observed. A small decrease in GSH peroxidase (GPx) mRNA abundance was observed in senescent cells. No other changes in the activities or mRNA abundances of any of the antioxidant defenses were observed in early and late passage cultures. The relationships between oxidant generation, mitochondrial enzyme activities, and antioxidant defense observed during proliferative senescence are dissimilar to those detected between fetal and postnatal fibroblasts as well as those found between fibroblast lines obtained from young and old individuals. The relevance of the differences between these models is discussed. J. Cell. Physiol. 180:114–122, 1999. © 1999 Wiley-Liss, Inc.     

Thymidine Kinase from Normal, Simian Virus 40-Transformed and Simian Virus 40-Lytically Infected Cells

Simian virus 40 (SV40) infection of human diploid cells failed to cause an enhanced production of thymidine kinase during the first 10 days after infection. Thymidine kinase activities from extracts of SV40-transformed cultures (human or simian) were considerably higher than the activity levels in extracts from the normal cells of origin. In addition, whereas the kinase activities obtained for human diploid cultures decreased as the cell sheet became confluent, the kinase activities for SV40-transformed human cells remained high after confluence was reached. Antisera obtained from hamsters bearing SV40 or adeno-7-SV40 hybrid virus tumors selectively inhibited enzyme from transformed sources (human or simian). Also, the antisera selectively inhibited enzyme extracted from SV40-lytically infected monkey cells. Sera from normal animals or from hamsters bearing polyoma tumors failed to inhibit enzymes from normal, SV40-transformed, or SV40-lytically infected cells. The Michaelis constant of partially purified enzyme from SV40-transformed cells was two to five times as high as that obtained for partially purified enzyme from human diploid cell cultures.     

Entry into S phase is inhibited in two immortal cell lines fused to senescent human diploid cells.

Senescent human diploid cells (HDC) were fused to T98G human glioblastoma cells and to RK13 rabbit kidney cells, and DNA synthesis was analyzed in the heterodikaryons. T98G and RK13 cells are “partially transformed” cell lines that have some characteristics of normal cells, yet are transformed to immortality, i.e., they do not senesce. Previous experiments have shown that “fully transformed” HeLa and SV80 cells induce DNA synthesis in senescent HDC nuclei, whereas normal young HDC do not. Our experiments show that T98G and RK13 cells do not induce DNA synthesis in senescent HDC nuclei. These results demonstrate that the ability to induce DNA synthesis in senescent HDC is not correlated with immortality per se. Our results show further that a T98G cell in S phase at the time of fusion to a senescent HDC will continue to make DNA. However, a T98G cell in G1 phase at the time of fusion is prevented from initiating DNA synthesis. RK13 cells behave similarly to T98G. These results are consistent with the hypothesis that the molecular basis for the senescent phenotype involves a block that prevents cells in G1 phase from entering S phase. Thus, we conclude that the senescent phenotype can be dominant in heterokaryons composed of senescent HDC fused with certain immortal cell lines. To explain the different results obtained with various immortal cell lines, we present a model that suggests that T98G and RK13 cells are immortal because they have lost a normal regulatory factor, whereas HeLa and SV80 are immortal because they have gained a dominant transformation factor.     

Transforming viruses directly reduce the cellular growth requirement for a platelet derived growth factor

Early passage mouse embryo fibroblasts, mouse 3T3 cell lines, and early passage diploid human fibroblasts grew to higher cell densities in tissue culture medium supplemented with serum than in medium supplemented with defibrinogenated platelet-poor plasma (PPP). Unlike the mouse cells, the human fibroblasts displayed this differential growth response only in the presence of hypophysiologic concentrations of calcium. The addition of heat-treated extracts of human platelets to PPP-supplemented medium stimulated the replication of both the normal mouse cells and early passage human embryo fibroblasts. Human or mouse fibroblasts transformed by either retroviruses or by SV40, including SV40 infected “serum revertants” and “flat transformants,” grew to equal cell densities in medium supplemented with either serum or PPP. Infection of Balb/c-3T3 cells with SV40 rapidly induced them to grow in PPP-supplemented medium demonstrating that the ability of SV40-transformed cell lines to proliferate in PPP-supplemented medium does not arise from the cell culture selection procedures usually employed to obtain stable virus-transformed cell lines. 3T3 cells infected but not transformed by retroviruses do not replicate in PPP-supplemented medium demonstrating that reduction of the growth requirement for the platelet growth factor(s) by retroviruses is a transformation-specific response. Cell cultures that did not proliferate well in PPP-supplemented medium did not form tumors when inoculated into athymic nude mice. Many, although not all, of the lines which grew well in PPP medium were tumorigenic in nude mice. Together, these findings indicate that: (1) normal fibroblast-like cells display a growth requirement for factor(s) present in serum but not found in PPP; (2) this serum specific growth factor is derived from platelets; (3) a primary response to viral transforming genes is a reduction in the growth requirement for these platelet-derived factors; and (4) cells that have a reduced requirement for the platelet-derived growth factor are often tumorigenic.     

Isolation of ES-Like Lines of Common Voles of the Genus Microtus from Blastocysts and Germ Cells and as a Result of Fusion of Somatic Cells with Mouse Embryonic Stem Cells

Three and four independent cell lines with limited pluripotency were obtained from the inner cell mass cells of blastocysts and primordial germ cells of common voles, respectively. The results of cytogenetic analysis suggest that all these lines originated from the embryos of F₁ Microtus rossiaemeridionalis × M. arvalis males and had a great number of near-triploid cells already during the early passages. The cells of these lines, like those of the inner cell mass, were characterized by the alkaline phosphatase activity. Nine independent cell lines were obtained as a result of hybridization of the mouse embryonic stem cells and vole splenocytes: eight lines and one line from hybridization with the M. kirgisorum and M. rossiaemeridionalis splenocytes, respectively. The cells of these lines expressed some properties of embryonic stem lines had a chromosome complement similar to the sum of two initial diploid sets of the mouse and vole.     

Characterization of a bone-specific alkaline phosphatase in chick limb mesenchymal cell cultures

Previous morphometric and biochemical studies suggested that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. Others have shown that osteoblast expression is marked by an increase in bone-specific alkaline phosphatase activity. Our results indicate that chick limb mesenchymal cells develop alkaline phosphatase activity that is identical to that of the chick embryonic bone-specific isoenzyme. The alkaline phosphatase isozymes were partially purified from samples of chick intestine, liver, stage 38 embryonic limbs, and cultures of stage 24 limb mesenchymal cells. These tissues were separately extracted with butanol, acetone precipitated, redissolved, and passed over a DEAE-Sephacel ion-exchange column and ion-filtration column (Sephadex A-25). From the data obtained during this purification scheme, we conclude that the alkaline phosphatase from stage 38 limbs (bones) and Day 4 cultures are identical, and this activity is different from the enzyme purified from intestine and liver. The cell culture isozyme has an apparent K_m, heat lability, response to specific inhibitors, electrophoretic mobility, and molecular weight similar to those of bone-specific alkaline phosphatase. These observations support the view that osteoblastic progenitor cells are present in the stage 24 limb mesenchyme and that under specific culture conditions, bone development can be uniquely observed in vitro.     

gamma-Glutamyl transpeptidase and glutathione in aging IMR-90 fibroblasts and in differentiating 3T3 L1 preadipocytes

γ-Glutamyl transpeptidase, glutathionase, and phosphate-independent glutaminase activities, and glutathione contents were measured in IMR-90 cells, human embryonic lung diploid fibroblasts, “aging” in culture, and in cultures of 3T3 L1 mouse preadipocytes differentiating to adipocytes. γ-Glutamyl transpeptidase, glutathionase, and phosphateindependent glutaminase activities in IMR-90 cells were about 8-, 8-, and 7-fold higher in “old” cells as compared to the activities in “young” cells. Total and reduced glutathione contents, expressed in relation to either cell number or DNA content, of IMR-90 cells were 3- and 12-fold higher in “young” cells (population doubling level 22) as compared to “old” cells (population doubling level 49). The ratio of reduced to oxidized glutathione was relatively constant in “young” cells over a range of population doubling levels 18–25; however, relative to the “young” cells, the ratio was decreased in “old” cells over a range of population doubling levels 49–55. γ-Glutamyl transpeptidase, glutathionase, and phosphate-independent glutaminase activities in 3T3 L1 cells were about 4-, 5-, and 5-fold higher in differentiating cells as compared to the activities in undifferentiating cells. Total glutathione contents in 3T3 L1 cells differentiating to adipocytes were similar to glutathione contents in undifferentiating cells. As the culture differentiated to the extent of 50%, almost all of the glutathione was present as oxidized glutathione. These results suggest that the several enzymatic activities and glutathione contents may be used as markers for aging or differentiation in cells. In the course of these studies, a sensitive fluorometric assay was developed, using l-γ-glutamyl-4-methoxy-2-naphthylamide as substrate.     

5'-Nucleotide phosphodiesterase and alkaline phosphatase in tumor cells: evidence for existence of novel species in the cytosol

Characteristics of 5'-nucleotide phosphodiesterase (phosphodiesterase I, EC 3.1.4.1) and alkaline phosphatase (EC 3.1.3.1) activities in tumor cell lines of human and murine origin were examined. Of the 15 cell lines tested, 5'-nucleotide phosphodiesterase activity in 13 cell lines and alkaline phosphatase activity in 10 cell lines were inhibited by N-ethylmaleimide and activated by dithiothreitol (N-ethylmaleimide-sensitive), and suggested to be SH-enzymes. In contrast, the two phosphohydrolases from normal tissues were inactivated by dithiothreitol, but not by N-ethylmaleimide (dithiothreitol-sensitive). There was only one tumor cell line in which both activities were dithiothreitol-sensitive. Human hepatoma PLC/PRF/5 cells appear to possess both types of 5'-nucleotide phosphodiesterase and alkaline phosphatase, and the subcellular distribution of these enzymes in this cell line was investigated. Dithiothreitol-sensitive 5'-nucleotide phosphodiesterase and alkaline phosphatase of PLC/PRF/5 cells were localized in the plasma membrane as in normal tissues, but N-ethylmaleimide-sensitive phosphohydrolases were soluble cytosolic proteins. N-Ethylmaleimide-sensitive 5'-nucleotide phosphodiesterase and alkaline phosphatase activities from other cell lines were also recovered in the cytosol. Molecular masses of cytosolic N-ethylmaleimide-sensitive phosphohydrolases were apparently smaller than their membrane-bound dithiothreitol-sensitive counterparts, as judged from gel filtration. It was concluded that many tumor cell lines lack plasma membrane 5'-nucleotide phosphodiesterase and alkaline phosphatase, but express enzymes with similar activities in the cytosol, with properties clearly distinguishable from enzymes so far characterized.     

Synthesis of alpha subunit of human chorionic gonadotrophin by presumptive HeLa cells

Summary Several cell lines, originally thought to be derived from a human placenta at term but possibly HeLa-contaminated, have been studied. These cells secrete a protein indistinguishable immunochemically from the alpha subunit of chorionic gonadotropin but not the beta subunit of chorionic gonadotropin or placental lactogen. Complete chorionic gonadotropin was detected but amounted to less than 1% of the level of the alpha subunit. The cells also produce an alkaline phosphatase similar to placental alkaline phosphatase in immunochemical, gel-electrophoretic, and heat-denaturation properties. They induce tumor growth when inoculated into nude mice. These cells are aneuploid and have a model chromosome number of 66. The common HeLa karyologic markers, designated 1, 2, and 3, and A-type glucose-6-phosphate dehydrogenase are present in these cells. HeLa cells have not previously been shown to secrete theα subunit of hCG.     

Increased alkaline phophatase activity in HeLa cells mediated by aliphatic monocarboxylates and inhibitors of DNA synthesis

Alkaline phosphatese activity of HeLa cells is increased from 3- to 8-fold during growth in medium with certain aliphatic monocarboxylates. The four-carbon fatty acid salt, sodium butyrate, is the most effective “inducer” with propionate (C₃), pentanoate (C₅) and hexanoate (C₆) having lesser effects. Other straight-chain aliphatic monocarboxylates, branched-chain analogues of inducers, hydroxylated derivatives, and metabolytes structurally related to butyrate are ineffective in mediating an increase in enzyme activity, indicating stringent structural requirements for inducers. The kinetics of increase in alkaline phosphatase activity in HeLa cells shows a 20–30 h lag period after adding the aliphatic acid followed by a rapid linear increase of enzyme activity. Protein synthesis is required for “induction”. The isozyme of HeLa alkaline phosphatase induced by monocarboxylates is the carcinoplacental form of the enzyme as determined by stereospecific inhibition by the l-enantiomorphs of phenylalanine and tryptophan, heat stability, and immunoreactivity with antibody against the human placental enzyme.Monocarboxylates that mediate increased alkaline phosphatase activity inhibit HeLa cell multiplication. Inhibition of HeLa cell growth may be necessary for induction and this hypothesis is supported by the findings that three different inhibitors of DNA synthesis, i.e. hydroxyurea, 1-β-d-arabinfuranosyl cytosine and methotrexate, also increase alkaline phosphatase activity. These inhibitors are synergistic with butyrate in causing HeLa cells to assume a more spindle-like shape and in producing an up-to 25-fold increase of enzyme activity. Studies on the modulation of carcinoplacental alkaline phosphatase by monocarboxylates commonly used as antimicrobial food additives and by anti-neoplastic agents may provide methods to evoke “tumor markers” of human occult malignancies. These drug-induced elevations of fetal isozyme activity may further our understanding of gene expression in human cells.     

Establishment of immortalized alveolar type II epithelial cell lines from adult rats

Summary We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37° C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham’s F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number. However, at later passages the 6T cells became polyploid, while the 6TN genotype remained stable. The RLE-6T and 6TN cells were not tumorigenic in nude mice. The cell isolation methods reported and the novel cell lines produced represent potentially useful tools to study the role of pulmonary epithelial cells in neoplastic and nonneoplastic lung disease.     

SV40-transformed human fibroblasts: evidence for cellular aging in pre-crisis cells

Pre-crisis SV40-transformed human diploid fibroblast (HDF) cultures have a finite proliferative lifespan, but they do not enter a viable senescent state at end of lifespan. Little is known about either the mechanism for this finite lifespan in SV40-transformed HDF or its relationship to finite lifespan in normal HDF. Recently we proposed that in normal HDF the phenomena of finite lifespan and arrest in a viable senescent state depend on two separate processes: 1) an age-related decrease in the ability of the cells to recognize or respond to serum and/or other mitogens such that the cells become functionally mitogen-deprived at the end of lifespan; and 2) the ability of the cells to enter a viable, G1-arrested state whenever they experience mitogen deprivation. In this paper, data are presented that suggest that pre-crisis SV40-transformed HDF retain the first process described above, but lack the second process. It is shown that SV40-transformed HDF have a progressively decreasing ability to respond to serum as they age, but they continue to traverse the cell cycle at the end of lifespan. Concomitantly, the rate of cell death increases steadily toward the end of lifespan, thereby causing the total population to cease growing and ultimately to decline. Previous studies have shown that when SV40-transformed HDF are environmentally serum deprived, they likewise exhibit continued cell cycle traverse coupled with increased cell death. Thus, these results support the hypothesis that pre-crisis SV40-transformed HDF still undergo the same aging process as do normal HDF, but they end their lifespan in crisis rather than in the normal G1-arrested senescent state because they have lost their ability to enter a viable, G1-arrested state in response to mitogen deprivation.     

Serum Lactic Dehydrogenase,Leucine Aminopeptidase and 5-Nucleotidase Activity: Observations in Patients with Carcinoma of the Pancreas and Hepatobiliary Disease

Serum lactic dehydrogenase, leucine aminopeptidase, 5-nucleotidase and alkaline phosphatase activities were investigated in a number of diseases involving the hepatobiliary system.Leucine aminopeptidase was found to be a sensitive indicator of biliary obstruction, serum 5-nucleotidase slightly less sensitive, and alkaline phosphatase appreciably less sensitive. Leucine aminopeptidase and 5-nucleotidase activities were often increased by malignant infiltration of the liver and primary hepatic disease even in the absence of jaundice.Serum lactic dehydrogenase was frequently increased in primary hepatic disease and malignant disorders but was not apparently affected by bile duct obstruction per se. Thirty-five of 45 patients with proved malignancy had increased lactic dehydrogenase levels.The highest leucine aminopeptidase levels were encountered in carcinoma of the head of the pancreas. The frequent increase in both serum lactic dehydrogenase and leucine aminopeptidase activities in patients with carcinoma of the head of the pancreas suggests that these combined estimations are useful laboratory procedures in the diagnosis of malignant extrahepatic obstruction.     

DNA polymerase α and the regulation of entry into S phase in heterokaryons

We have previously reported that the DNA polymerase α activity/unit cellular protein is decreased in latepassage (senescent) human diploid fibroblast-like (HDFL) cultures due to the cellular enlargement associated with in vitro aging. In the studies described here, we have used cell fusion technology to investigate the formal kinetic relationship between the concentration of DNA polymerase α and the rate of reinitiation of DNA synthesis in nuclei from senescent cells. Heterokaryons were derived from the fusion of senescent cells to a series of actively dividing cell types with inherently different DNA polymerase α activities per cell. A kinetic analysis revealed a first-order relationship between the entry into S phase of senescent nuclei and the concentration of DNA polymerase a activity calculated to be in heterokaryons. This result suggests that increases in cell volume may be related to the decline in proliferative activity of late-passage HDFL cells, via “dilution” of factors essential for cellular replication.     

Cystic fibrosis: leakage of lysosomal enzymes and of alkaline phosphatase into the extracellular space

Nine lysosomal enzymes and alkaline phosphatase have been assayed with two different ultramicro techniques in the intra- and extracellular space of fibroblast cultures derived from the skin of cystic fibrosis patients, cystic fibrosis carriers, and normal controls, respectively. Evidence has been obtained for a multiple leakage of lysosomal enzymes and of alkaline phosphatase into the medium of fibroblast cultures from cystic fibrosis patients and carriers. The situation is comparable to a certain extent, to that observed in I-cell-disease (mucolipidosis II). This multiple leakage results in the decrease of intracellular activity of several lysosomal enzymes in cultures from cystic fibrosis patients and carriers and due to the coordinate regulation of the synthesis of the “leaky enzymes” in an overshooting of the intracellular alkaline phosphatase activity in cultures from cystic fibrosis patients. It also explains the retarded catabolism of certain molecules, such as the Tamm-Horsfall glycoprotein, in cystic fibrosis cells. It is speculated that the basic defect in cystic fibrosis leads to abnormal recognition sites on lysosomal enzymes and on alkaline phosphatase, and in consequence to the leakage of these enzymes into the extracellular space. The present findings allow one to develop methods for the pre- and postnatal diagnosis of cystic fibrosis with cell cultures, and for the detection of cystic fibrosis carriers with the peripheral blood.     

DNA polymerase alpha and the regulation of entry into S phase in heterokaryons

We have previously reported that the DNA polymerase alpha activity/unit cellular protein is decreased in late-passage (senescent) human diploid fibroblast-like (HDFL) cultures due to the cellular enlargement associated with in vitro aging. In the studies described here, we have used cell fusion technology to investigate the formal kinetic relationship between the concentration of DNA polymerase alpha and the rate of reinitiation of DNA synthesis in nuclei from senescent cells. Heterokaryons were derived from the fusion of senescent cells to a series of actively dividing cell types with inherently different DNA polymerase alpha activities per cell. A kinetic analysis revealed a first-order relationship between the entry into S phase of senescent nuclei and the concentration of DNA polymerase alpha activity calculated to be in heterokaryons. This result suggests that increases in cell volume may be related to the decline in proliferative activity of late-passage HDFL cells, via "dilution" of factors essential for cellular replication.