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J J Freed

Publications and source records attributed to J J Freed.

At least 19 recordsLinked to original sources
PubMed1995

Genomic alterations and instabilities in renal cell carcinomas and their relationship to tumor pathology.

A comprehensive genome scan for loss of heterozygosity (LOH) in 33 renal cell carcinomas indicates that mutations of tumor suppressor genes on several different chromosomes are required for malignant transformation in this disease. In the case of nonpapillary renal carcinomas chromosomes 3p, 6q, 8p, 9pq, and 14q exhibit elevated levels of LOH. Although 3p is the most frequently lost chromosome arm, in no case is 3p observed as the sole allelic loss because it always occurs in conjunction with the loss of either 6q, 8p, or 14q. This result indicates that the mutation of a tumor suppressor gene on 3p, most likely von Hippel-Lindau disease (VHL), may be necessary but is not sufficient for the development of nonpapillary renal cell carcinoma. In papillary renal tumors, LOH is observed most often for chromosomes 6pq, 9p, 11q, 14q, and 21q. This suggests that tumor suppressor genes located on chromosomes 6q, 9pq, and 14q may be involved in the development and/or progression of both nonpapillary and papillary renal cell carcinomas. However, LOH in papillary tumors appears to be especially elevated for 11q and 21q and reduced for 3p and 8p indicating that there are also tumor suppressor genes specific to each form of the disease. There is no correlation between stage of disease and the extent of LOH, loss of a particular chromosome, or the number of chromosomes that show allele imbalance. Early and late stage tumors may exhibit either extensive LOH or no apparent allele loss; similarly, allelic imbalances are observed in both early and late stage renal cell carcinomas. This suggests that a gene (or genes) regulating mitotic chromosome stability may be mutated in some renal tumors. Preliminary evidence points to an association between genome instability and LOH of 14q. Finally, a distinct type of microsatellite instability has been detected in 21% of renal cell carcinomas and occurs at a frequency of 4.4 x 10(-4)/locus. The most common mutation is a 2-bp insertion in a CA repeat. This alteration is not restricted to a particular histopathology or clinical stage, and it is not associated with allelic loss of a specific chromosome. The frequency of this event is similar to that which occurs spontaneously in germline microsatellite loci and is probably not the result of a defect in a mismatch repair gene. It is possible that this type of microsatellite instability is general and may occur in most, if not all, carcinomas.

Alleles↗
PubMed1995

Comprehensive allelotyping of human renal cell carcinomas using microsatellite DNA probes.

The von Hippel-Lindau locus on chromosome 3p is a tumor suppressor gene known to be involved in nonpapillary renal cell carcinoma. A previous loss of heterozygosity (LOH) study aimed at determining the allelotype of kidney tumors has indicated that in addition to 3p, chromosome arms 5q, 6q, 10q, 11q, 17p, and 19p may also harbor tumor suppressor genes. However, cytogenetic studies reveal that chromosomes 3p, 6q, 8p, 9pq, and 14q most frequently undergo karyotypic changes in renal tumors. To resolve these differences, a collection of microsatellite DNA probes has been used to scan for LOH so that 90% of individual tumor genomes were rendered informative for allele loss. The assay is capable of detecting quantitative genomic alterations in tumor cells as well. We find that LOH is most frequent for chromosome arm 3p. However, in no tumor is 3p exclusively affected. LOH for 6q, 8p, 9pq, and 14q is also distinctly elevated for both nonpapillary as well as papillary tumors and suggest that many of the tumor suppressor loci involved may be common to the etiology of both forms of kidney cancer.

Alleles↗
PubMed1993

Spontaneous and radiation-induced renal tumors in the Eker rat model of dominantly inherited cancer.

Hereditary renal carcinoma (RC) in the rat, originally reported by R. Eker in 1954, is an example of a Mendelian dominant predisposition to a specific cancer in an experimental animal. At the histologic level, RCs develop through multiple stages from early preneoplastic lesions (e.g., atypical tubules) to adenomas in virtually all heterozygotes by the age of 1 year. The homozygous mutant condition is lethal at approximately 10 days of fetal life. Ionizing radiation induces additional tumors in a linear dose-response relationship, suggesting that in heterozygotes two events (one inherited, one somatic) are necessary to produce tumors, and that the predisposing gene is a tumor suppressor gene. No genetic linkage has yet been found between the Eker mutation and rat DNA sequences homologous to those in human chromosome 3p, the presumed site of the putative tumor suppressor gene responsible for human RC. Nonrandom loss of rat chromosome 5 in RC-derived cell lines is sometimes associated with homozygous deletion of the interferon gene loci at rat chromosome bands 5q31-q33. Since this locus is not linked with the predisposing inherited gene in the Eker rat, it probably represents a second tumor suppressor gene involved in tumor progression.

Adenoma↗
PubMed1991

Hereditary renal cell carcinoma in the rat associated with nonrandom loss of chromosomes 5 and 6.

A spontaneous form of renal cell carcinoma occurs in rats that arises as the result of the inheritance of a mutation in a single autosomal gene. Cytogenetic analysis was performed on seven cell lines and four primary tumor cell preparations derived from this hereditary form of renal cell carcinoma. Banded karyotypes prepared from these seven lines exhibited loss and/or partial deletion of both chromosomes 5 and 6. Translocations involving chromosome 4, resulting in a net loss of genetic material located near the centromere (4q11), were observed in three of the cell lines. Monosomy, translocation, and breakage of chromosome 5 involving band 5q31 and monosomy and partial deletion of chromosome 6 involving band 6q22-q24 were independently observed in primary tumor cells from three of four tumors examined. Monosomy of chromosome 4 was observed in cells from a single tumor. The smallest region of deletion of chromosome 6 common to all the cell lines and tumor cells was 6q24, suggesting the presence of a tumor suppressor gene at this locus. These results indicate that loss of genes located on chromosomes 4, 5, and 6, possibly tumor suppressor gene(s), may be important for tumor development and/or progression in rat renal cell carcinoma and is consistent with the hypothesis that a gene locus on one of these chromosomes may be the site of the original predisposing mutation.

Animals↗
PubMed1991

Altered expression of transforming growth factor-alpha in hereditary rat renal cell carcinoma.

A hereditary form of renal cell carcinoma exists in rats that results from a single gene mutation and is histologically similar to that described in humans. Cell lines derived from these rat tumors were shown to express abundant transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF)-receptor RNA transcripts, but no EGF mRNA. In contrast, normal kidney expressed EGF and EGF-receptor transcripts, but TGF-alpha transcripts were barely detectable. Other kidney epithelial cell lines examined (NRK 52E, MDCK, and LLCPK) were negative for expression of both TGF-alpha and EGF transcripts, but expressed EGF receptors. In addition, the renal cell carcinoma-derived lines secreted TGF-alpha into the media. Immunohistochemistry of normal kidney with a TGF-alpha specific antibody revealed a characteristic pattern of staining of collecting ducts and, to a lesser degree, proximal tubules. In the neoplastic kidney tissue, both the cystic and solid portions of the tumors displayed intense immunoreactivity, indicating that altered expression of this growth factor by the transformed cells occurred in situ. These results suggest that altered TGF-alpha expression is an important aspect of the neoplastic phenotype in rodent as well as human renal cell carcinoma, and support the use of this hereditary rodent tumor model for studying the pathogenesis of this disease.

Animals↗
PubMed1989

Evidence for acyloxymethyl esters of pyrimidine 5'-deoxyribonucleotides as extracellular sources of active 5'-deoxyribonucleotides in cultured cells.

Cells commonly resist growth inhibition by purine and pyrimidine bases and nucleosides by restricting intracellular formation of the corresponding 5'-mononucleotides. Nucleotide derivatives that can act as effective membrane-transport precursors of the poorly membrane-permeable nucleotides have not been identified so far. We studied the bis(pivaloyloxymethyl)ester (I) of FdUMP (5-fluoro-dUMP) and a cyclic phosphodiester (II) of FdUMP derived from 1,3-dihydroxyl-1-C-(pivaloyloxy-methyl)propane which are active in vivo against a 5-fluoro-2'-deoxyuridine (FUdR)-resistant mouse leukemia and are attacked by carboxylic esterases under physiological conditions to produce FdUMP by elimination of formaldehyde and acrolein respectively. The assay for intracellular FdUMP was the inhibition of DNA synthesis due to inhibition of TMP synthetase in cultured mouse LM(TK-) fibroblasts genetically devoid of thymidine kinase (TK) and thus unable to convert FUdR directly to FdUMP. At 10(-6)M, I, II, or FUdR inhibited DNA synthesis in 2 hr by 99, 80, and 35% respectively; at 10(-5)M. maximal inhibition was attained after less than 15, 30 and 90 min respectively. Inhibition of DNA synthesis in TK+ cells by 10(-5) M I, II, or FUdR was reversed completely by 10(-5)M thymidine (TdR) but unaffected by 10(-5)M UdR, confirming TMP synthetase as the locus of inhibition. At 10(-5)M, bis(pivaloyloxymethyl) esters of phenyl phosphate or a p-substituted benzylphosphonic acid did not inhibit significantly DNA synthesis in TK+ cells. From this finding, and from effects produced by V (see below), we conclude that pivalic acid and CH2O arising from I contribute little to its above inhibitory effects. In TK- cells in which DNA synthesis is prevented by blockade of TMP synthetase with aminopterin, the bis(pivaloyloxymethyl) ester (V) of TMP, at 0.9 x 10(-4) M, induced a 4-fold faster rate of DNA synthesis than did 10(-3)M TMP, whereas 10(-3) M TdR did not affect the rate. After 3 hr the rate with V was 80% that in the absence of aminopterin. In the above systems the nucleotide diesters I, II and V appear to be acting as effective extracellular sources of active intracellular FdUMP and TMP, in processes that involve loss of the two esterifying groups.

Animals↗
PubMed1989

Bicarbonate abolishes intracellular alkalinization in mitogen-stimulated 3T3 cells.

An increase in intracellular pH (pHi) following mitogenic stimulation has been reported in a variety of mammalian cells (W. Moolenaar, Annu. Rev. Physiol., 48:363-376, 1986; E. Rozengurt, Science, 234:161-166, 1986). This increase is currently believed to constitute a "permissive" signal in the process of cell activation (A.E. Lagarde and J.M. Pouyssegur, Cancer Biochem. Biophys. 9:1-14, 1986). Since the majority of studies of this phenomenon have been conducted in the nonphysiological milieu of bicarbonate-free solutions, we have undertaken a study of the effects of bicarbonate and CO2 on mitogen-induced intracellular alkalinization in NIH 3T3 cells. Using nuclear magnetic resonance (NMR) spectroscopy and novel 31P NMR pH indicators (2-amino-phosphono-carboxylic acids) we found that mitogen induces an increase in pHi of 0.16 units only in cells bathed in medium containing low concentrations of bicarbonate (less than 1 mM) and not in cells bathed in medium containing physiological levels of bicarbonate (10-30 mM). In addition to abolishing the mitogen-induced alkalinization, bicarbonate stabilizes pHi at 7.25 units as the external pH (pHe) is varied from 7.0 to 7.6. In contrast, in a bicarbonate-free medium pHi increases from 6.9 to 7.3 over the same range of external pHs. At a constant external pH, increasing the bicarbonate/CO2 concentration results in an increase in pHi from 6.9 in bicarbonate-free solution to 7.25 in a bicarbonate-buffered medium. This relationship is hyperbolic with half-maximal effect occurring at a concentration of 0.4 mM bicarbonate at pH 7.05 and 37 degrees C. Our results suggest that the observations of mitogen-induced alkalinization may be due to the use of nonphysiological bicarbonate-free media. Since this increase in pHi is not observed in physiological media where bicarbonate concentrations are usually greater than 20 mM, we conclude that an increase in pHi is not an obligatory or usual part of the cellular response to growth factors in vivo.

Animals↗
PubMed1989

Expression of asparagine independence in variants of ICR 2A haploid frog cells.

Properties of the change from asparagine dependence (asn-) to independence (asn+) were investigated in the androgenetic haploid frog cell line ICR 2A. Two types of asn+ variants arose spontaneously during culture. Glutamine-dependent asparagine synthetase (AS) activity, found to be deficient in asn- cells, was repressed by asparagine in one type of variant and expressed constitutively in the other. No quantitative differences in AS-specific DNA sequences or changes in ploidy were evident between asn+ and asn- cells. The asn+ frequency in ICR 2A populations, not dramatically influenced by chemical mutagens, was increased 130-fold by exposure to 5-azacytidine. The methylation of CCGG sequences at the 5' end of the AS structural gene was found to be reduced equally in both types of asn+ variant. These results indicate that decreased DNA methylation is essential but not necessarily sufficient for the expression of AS activity in this frog cell system.

Animals↗
PubMed1988

Regional mapping of unique DNA sequences from human chromosome 3 derived from a flow-sorted chromosome library.

Eight single-copy DNA probes specific for human chromosome 3 were isolated by screening a human chromosome 3-derived genomic library. Southern blot analyses of DNAs isolated from a panel of somatic cell hybrids allowed us to regionally assign all probes to subregions on chromosome 3. Three clones were localized to the short arm of chromosome 3 (3p21----pter), two to the long arm (3q21----qter), and three to the 3q21----3p21 subregion. Six of these DNA sequences map to regions overlapping a segment of chromosome 3 (3p14----p23) frequently deleted in small cell lung cancer cells. Restriction fragment length polymorphism analyses indicate that at least three of the eight single-copy probes studies show MspI or BglII polymorphisms. This library is a useful source of chromosome 3-specific probes.

Chromosome Mapping↗
PubMed1986

Zwitterionic 3'-O-acyl derivatives of thymidine 5'-phosphate as potential sources of intracellular thymidine 5'-phosphate in cells in culture.

A convenient route is described for attachment of acyl groups CO(CH2)nN(Et)2(CH2)mNH(Et)2 (n = 3, m = 2; n = 4, m = 2-4), CO(CH2)nN(Et)2(CH2)mNEt3 (n = 4, m = 2-4), or CO(CH2)4N(CH2CH2)3N(CH2)nCH3 (n = 1 or 9) to O-3' of thymidine 5'-phosphate (TMP). The compounds are prototypes of 5'-nucleotide derivatives in which the two anionic charges could become partially masked in intramolecular anionic-cationic interactions and which might be able to diffuse into mammalian cells to furnish intracellular antimetabolite 5'-nucleotides by hydrolytic loss of a dicationic 3'-O-acyl group. At pH 7.6, 37 degrees C, hydrolyses of the 3'-ester linkages were pseudo first order with t1/2 values in the range 28-85 h. Paper chromatography in n-PrOH-H2O at pH 7.6 showed that type 1 or 2 derivatives were equally or slightly less hydrophobic than TMP (Rf 0.24), whereas the n-decyl type 3 compound (Rf 0.66) was markedly more hydrophobic, apparently because chain branching in group 3 is less than in 1 or 2. A sensitive and specific assay was developed for liberation of intracellular TMP in cultured mouse L fibroblasts in which synthesis of TMP and, hence, of DNA was suppressed by a combination of aminopterin and 5'-amino-5'-deoxythymidine (5'-NH2-dT). The TMP derivatives (100 microM) stimulated DNA synthesis, but omission of 5'-NH2-dT increased stimulation 6.5-fold, suggesting that stimulation occurred via degradation of the derivatives to dT. In confirmation, derivatives of type 2 (n = 4, m = 2 or 4) (100 microM) or type 3 (n = 9) (200 microM), in the presence of aminopterin, did not stimulate DNA synthesis in the LM(TK-) strain of L cells, which is genetically deficient in dT kinase. TMP (1 mM) stimulated DNA synthesis 2-3-fold and appeared to enter LM(TK-) cells without dephosphorylation, because dT (1 mM) gave no stimulation. If TMP is assumed to enter solely by passive diffusion, the inactivity of the TMP derivatives can be ascribed in part to their 2-fold higher molecular weight which can be expected to reduce flux through natural membranes ca. 16-fold.

Acylation↗
PubMed1984

Bis(m-nitrophenyl) and bis(p-nitrophenyl) esters and the phosphorodiamidate of thymidine 5'-phosphate as potential sources of intracellular thymidine 5'-phosphate in mouse cells in culture.

Thymidine 5'-phosphate (TMP) derivatives with masked phosphate groups were synthesized in tritiated form from [methyl-3H]thymidine. They were of interest as models for 5' nucleotide derivatives that might be able to permeate mammalian cells and then liberate intracellular antimetabolite 5' nucleotides by loss of the masking groups. Mouse L fibroblasts were grown in vitro in the presence of 1 mM 5'-amino-5'-deoxythymidine, which was found to suppress greater than 99% of cellular thymidine kinase activity while inhibiting the rate of cell division by only 30%. The TMP derivatives were less effective than thymidine in labeling the deoxyribonucleic acid (DNA) of the L cells. The labeling was inhibited 95-99% by 5'-amino-5'-deoxythymidine, indicating that it represented incorporation into DNA of [3H]thymidine formed from degradation of the test compounds. No evidence was obtained that the compounds acted as sources of intracellular TMP by cell permeation followed by loss of phosphate blocking groups. Similar studies yielded no evidence that the bis(m-nitrophenyl) ester of TMP produced intracellular TMP by that route in the LM(TK-) strain of L cells that are genetically deficient in thymidine kinase.

Amides↗
PubMed1980

Investigation of the determinants of nuclear pore number.

To assess the functional significance of nuclear pore complexes, we have investigated whether the number of pores per nucleus is determined by such factors as the nuclear volume, nuclear surface area, DNA content, or aspects of nuclear activity. Comparisons were made between cell types chosen to permit observation of differences in nuclear pore number as a function of differences in the other qualities measured. The number of nuclear pores was determined by freeze-etching and measurements of nuclear surface and nuclear volume by electron and light microscopy. Pairs of cell strains in culture that contained different numbers of chromosome sets were investigated to examine the relation of pore number to total DNA content. Tetraploid cells of the rat kangaroo (Potorous tridactylus) have almost exactly twice the number of pores found in the parental diploid strain. However, the pore number in diploid grassfrog (Rana pipiens) cells was only 65% greater than in the parental haploid cells. In addition, a polyploid series of nucleated RBC had a 62% pore number increase with each successive increase in ploidy. Diploid cell strains from the canyon mouse (Peromyscus crinitus) and from the cactus mouse (P. eremicus) were compared to test whether a difference reflecting the 36% additional DNA in cells of the latter, associated with extra heterochromatin, existed. Although both were found to have the same number of pores and nuclear surface area, the cells differed in nuclear volume. These observations suggest that the number of nuclear pores is independent of the total amount of nuclear DNA, the nuclear surface area (and, thus, presumably the fraction of DNA that is bound to the nuclear membrane), the nuclear volume, and the size of the genome. Rather, the number of nuclear pores appears to be associated with some aspect of nuclear metabolic activity, e.g., transcriptional capacity or release of products to the cytoplasm. Further evidence for such a view comes from studies of chick embryo erythroblasts. In these, nuclear pore number was found to be lower in associated with the decreasing nuclear transcriptional activity and longer generation times that characterize the successive cell divisions leading to the fully differentiated state. The number of pore complexes reconstructed in the last cell cycles declined in a manner consistent with reutilization of previously formed pores in the absence of new pore synthesis. Challenging this interpretation is the increase in pore number at lower metabolic activity when Xenopus laevis cells are grown at different temperatures. The speculation that pore complexes have a longer half-life in cooler grown Xenopus cells could resolve the discrepancy.

Animals↗
PubMed1979

Survival and DNA repair in ultraviolet-irradiated haploid and diploid cultured frog cells.

Survival and repair of DNA following ultraviolet (254-nm) radiation have been investigated in ICR 2A, a cultured cell line from haploid embryos of the grassfrog, Rana pipiens. Survival curves from cells recovering in the dark gave mean lethal dose value (Do) in the range 1.5--1.7 Jm-2 for both haploid and diploid cell stocks. The only significant difference observed between haploids and diploids was in the extent of the shoulder at low fluence (Dq), the value for exponentially multiplying diploid cells (3.0 Jm-2) being higher than that found for haploids (1.2 Jm-2). Irradiation of cultures reversibly blocked in the G1 phase of the cell cycle gave survival-curve coefficients indistinguishable between haploids and diploids. Post-irradiation exposure to visible light restored colony-forming capacity and removed chromatographically estimated pyrimidine dimers from DNA at the same rates. After fluences killing 90% of the cells, complete restoration of survival was obtained after 60-min exposure to 500 foot-candles, indicating that in this range lethality is entirely photoreversible and therefore attributable to pyrimidine dimers in DNA. Dimer removal required illumination following ultraviolet exposure, intact cells and physiological temperature, implying that the photoreversal involved DNA photolyase activity. Excision-repair capacity was slight, since no loss of dimers could be detected chromatographically during up to 48 h incubation in the dark and since autoradiographically detected "unscheduled DNA synthesis" was limited to a 2-fold increase saturated at 10 Jm-2. These properties make ICR 2A frog cells useful to explore how DNA-repair pathways influence mutant yield.

Animals↗
PubMed1976

Selective isolation of reversible cold sensitive variants from Chinese hamster ovary cell cultures.

Variants of the Chinese hamster ovary cell line CHO-KI (ATTCCCL61)which grow almost normally at 38.5C but very poorly or not at all at 30C were obtained after treatment with mutagens and application of an indirect selection procedure. Two kinds of variants were recovered. In the first of these, the cold sensitive phenotypw is expressed completely only at low cell densities. At higher cell desity, growth continues at the nonpermissive temperature, but at a reduced rate. In the second class, the cold snesitive phenotype is independent of cell density. Two members of the latter class were studied in detail. In both lines, after shift to the nonpermissive temperature, the rateof H-thymidine incorporation declines markedly; the rates of H-uridine and H-phenylalanine uptake are less drastically reduced. Autoradiographs indicate that the decline in thymidine uptake at the nonpermissive temperature is due to an elongation of part of the cell cycle, so that a smaller proportion of the cells lie in the synthetic (S) phase of the cell cycle with a consequent reduction in the fraction labeled cells. The uridine labeling patterns of the mutants appear to rule out a ribsomal lesion. Low temperature growth inhibition of both cell strains was reversible. In one of the cell lines, an apparent stretching of the cells at the low temperature produces substantial alterations in cell shape.

Cell Division↗