A genome-wide linkage scan for genes controlling variation in urinary albumin excretion in type II diabetes.

The main hallmark of diabetic nephropathy is elevation in urinary albumin excretion. We performed a genome-wide linkage scan in 63 extended families with multiple members with type II diabetes. Urinary albumin excretion, measured as the albumin-to-creatinine ratio (ACR), was determined in 426 diabetic and 431 nondiabetic relatives who were genotyped for 383 markers. The data were analyzed using variance components linkage analysis. Heritability (h2) of ACR was significant in diabetic (h2=0.23, P=0.0007), and nondiabetic (h2=0.39, P=0.0001) relatives. There was no significant difference in genetic variance of ACR between diabetic and nondiabetic relatives (P=0.16), and the genetic correlation (rG=0.64) for ACR between these two groups was not different from 1 (P=0.12). These results suggested that similar genes contribute to variation in ACR in diabetic and nondiabetic relatives. This hypothesis was supported further by the linkage results. Support for linkage to ACR was suggestive in diabetic relatives and became significant in all relatives for chromosome 22q (logarithm of odds, LOD=3.7) and chromosome 7q (LOD=3.1). When analyses were restricted to 59 Caucasian families, support for linkage in all relatives increased and became significant for 5q (LOD=3.4). In conclusion, genes on chromosomes 22q, 5q and 7q may contribute to variation in urinary albumin excretion in diabetic and nondiabetic individuals.

Molecular events in radiation transformation.

Studies of human tumor cell lines have revealed alterations in the regulation of a number of cell cycle-related genes, associated in some cases with a TP53-independent loss of the radiation-induced G(1)-phase arrest. It is not clear, however, whether these are early or late events in tumor development, or they arise in tumor cell lines during growth in culture. Since the oncogenic transformation of an individual cell is thought to be an early event in tumor development, we have used a model system of normal and radiation-transformed C3H 10T(1/2) mouse fibroblast cell clones to address this issue. Transformed clones derived from type III foci were compared with clones derived from parental, wild-type cells. Approximately 25% of transformed clones showed Trp53 mutations in exon 5; however, preliminary results based on in situ immunofluorescence studies with an antibody recognizing mutant Trp53 indicate that the appearance of such mutations in transformed clones occurs late in the process of transformation and is unlikely to represent an initiating event. The remaining transformed clones and all clones derived from parental cells expressed wild-type Trp53. Radiation-induced G(1)-phase arrest was either absent or significantly reduced in all of the transformed clones, independent of Trp53 status. Constitutive expression of Cdkn1a protein was significantly increased in most of the transformed clones. Also, the majority of transformed clones showed elevated levels of cyclin D1, and two clones overexpressed cyclin E. These results indicate that loss of G(1)-phase checkpoint control, independent of Trp53 status, and altered expression of cell cycle regulatory proteins may represent early events in the process of radiation-induced carcinogenesis that are associated with the malignant transformation of individual cells.

Overexpression of p21 protein in radiation-transformed mouse 10T(1/2) cell clones.

In order to investigate the hypothesis that aberrant expression of cell-cycle regulatory proteins may represent early events in the process of carcinogenesis, levels of expression of the negative regulators p21(waf1/cip1) (p21), p27(kip1) (p27), and p16(ink4a) (p16) and/or the positive regulators cyclin D(1) and cyclin E were examined by western blot analysis in cells transformed in vitro by ionizing radiation. The levels of these proteins in 12 independently derived mouse 10T(1/2) cell clones transformed by 1.5 Gy of alpha radiation were compared with those in nine similarly derived nontransformed control clones. Constitutive levels of p21 were very low in all control clones, whereas p21 expression was significantly elevated in nine of 12 transformed clones. Two of the three transformed clones displaying low levels of p21 expressed increased levels of p53. p21 regulation was also altered in response to radiation in transformed clones as compared with controls, only minimal induction was observed 4 h following gamma irradiation. Western blot analysis indicated a constant expression of p27 protein but slightly decreased levels of p16 in these transformed clones. Cyclin D(1) was overexpressed in 11 of 12 transformed clones; in only two of these were the levels of cyclin E elevated. Overall, the results suggest that alterations in the expression of cell cycle regulatory proteins may represent important events in radiation-induced oncogenic transformation in vitro. Although the specific alterations vary among different transformed clones, overexpression and aberrant regulation of p21 appear to be the most frequent ones.

Loss of normal G1 checkpoint control is an early step in carcinogenesis, independent of p53 status.

Recent studies have described a diminished radiation-induced G1 arrest in some wild-type (wt) p53 human tumor cell lines compared to normal human fibroblasts. However, the significance of this finding was unclear, particularly because tumor cell lines may have accumulated additional genetic changes after long periods in culture. Because malignant transformation of individual cells is thought to be an early step in carcinogenesis, we have used a model system of normal and transformed mouse fibroblast 10T1/2 cell clones to examine whether loss of G1 checkpoint control may be an early event in tumor development and to study the relationships between G1 arrest, radiosensitivity, and genetic alterations. Twelve transformed clones were established from type III foci induced by irradiation of normal 10T1/2 cells and were compared with six clones derived from wt 10T1/2 cells. Three of the transformed clones expressed mutant p53; two of these had the same point mutation at codon 132 (exon 5), and one had a point mutation at codon 135. The remaining transformed and normal clones had wt p53 status. The radiosensitivity of transformed clones, as measured by a clonogenic assay, was similar to that of normal clones; the three clones with mutant p53 did not differ from the others. There was no relationship between G1 arrest and radiosensitivity. Normal 10T1/2 cell clones showed a transient G1 arrest lasting approximately 9 h after 6 Gy of irradiation. This G1 arrest was either absent or markedly reduced in all of the transformed clones, regardless of p53 status. These results suggest that diminished G1 checkpoint control is an early event in the process of carcinogenesis that is associated with the malignant transformation of individual cells and is independent of p53 status.

Phosphorylation and activity of Na+/H+ exchanger isoform 1 of immortalized lymphoblasts in diabetic nephropathy.

In both essential hypertension and diabetic nephropathy (DN), the ubiquitous cellular Na+/H+ exchanger (NHE) exhibits altered kinetics with increased transport activity. The mechanism for this phenotype and its dependence on the presence of serum are unknown, but increased lymphoblast NHE activity in DN has been attributed to a defect in post-translational processing of NHE-1 rather than an increased cellular exchanger number. Phosphorylation of NHE-1 has been proposed to play a role in its activation in a variety of cell models. We have examined, therefore, the role of NHE-1 phosphorylation and the effect of serum in determining the increased NHE-1 activity in lymphoblasts from patients with DN. Cells from these patients exhibited increased NHE activity in the presence and absence of fetal calf serum (range 42-59%, P < 0.005, analysis of variance) and an increased proliferation rate (P < 0.01) when compared with cells from both normoalbuminuric diabetic patients and non-diabetic control subjects. However, NHE-1 abundance was very similar among all groups in the presence and absence of serum, indicating that increased NHE activity in cells of nephropathy patients was due to an increased turnover number. This nephropathy phenotype was not accompanied by an increased net phosphorylation of NHE-1 in the presence or absence of serum. Our findings suggest that increased NHE-1 activity in cells of DN patients is independent of the presence of serum and is not attributable to altered NHE-1 phosphorylation. Additional post-translational mechanisms for activation of NHE-1, therefore, may be involved.

Na(+)-H+ antiporter phenotype, abundance, and phosphorylation of immortalized lymphoblasts from humans with hypertension.

Previous studies have demonstrated an elevated Na(+)-H+ exchanger activity in various cell types from patients with essential hypertension. The phenotype of an increased maximal transport capacity is preserved in Epstein-Barr virus immortalized lymphoblasts from hypertensive patients. The mechanisms underlying this abnormality are unclear. In this study, we used lymphoblasts from hypertensive patients and normotensive control subjects with and without a family history of hypertension to determine (1) Na(+)-H+ exchanger activity using fluorometry with the pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, (2) Na(+)-H+ exchanger isoform 1 abundance with specific polyclonal antibodies, and (3) Na(+)-H+ exchanger phosphorylation by immunoprecipitation of the 32P-labeled transporter. Na(+)-H+ exchanger activity (in millimoles per liter per minute) measured when pHi was clamped at 6.0 was significantly higher in cells from hypertensive patients (18.8 +/- 0.6, P < .001) and those subjects with a family history of hypertension (16.4 +/- 0.6, P < .001) compared with normotensive control subjects (12.9 +/- 0.6). Exchanger abundance was identical in all three groups of subjects, indicating that increased activity in the hypertensive group was due to an elevated turnover number of the exchanger. Na(+)-H+ exchanger phosphorylation in quiescent cells was significantly elevated in cells from hypertensive patients (1.58 +/- 0.16, P < .001) compared with control subjects (1.00 +/- 0.07), and cells from normotensive subjects with a hypertensive family history showed intermediate values (1.23 +/- 0.14). Identical changes in Na(+)-H+ exchanger function and phosphorylation have been demonstrated in vascular smooth muscle cells from spontaneously hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose-induced changes in turnover of Na+/H+ exchanger of immortalized lymphoblasts from type I diabetic patients with nephropathy.

Increased cellular Na+/H+ exchanger (NHE) activity has been demonstrated in type I diabetic patients with nephropathy. Such patients also have a previous history of poor glycemic control. The interaction between hyperglycemia and changes in NHE activity remains obscure. Therefore, we examined the effects of media containing 5 and 25 mmol/l glucose on the increased NHE activity and turnover number in Epstein-Barr virus-transformed lymphoblasts from patients with diabetic nephropathy compared with normoalbuminuric diabetic and nondiabetic control subjects. NHE activity was determined fluorometrically, and NHE isoform 1 (NHE-1) density was measured with specific polyclonal antibodies. In the presence of 5 mmol/l glucose, cells from patients with diabetic nephropathy exhibited higher NHE activity with intracellular pH clamped to 6.0 compared with diabetic and nondiabetic control subjects (P < 0.005 for both), due to a higher turnover number of NHE-1. Incubation in 25 mmol/l glucose for 48 h caused an increase in NHE activity (P < 0.001) and turnover number (P < 0.01) in the diabetic nephropathy group only, with no significant change in the diabetic or nondiabetic control groups. The rate constants for cell proliferation and NHE activity or turnover number were correlated when cells were cultured in 5 mmol/l glucose (r = 0.34 and 0.32, respectively; P < 0.05) or 25 mmol/l glucose media (r = 0.66 and 0.65, respectively; P < 0.001). We conclude that only lymphoblasts from the diabetic nephropathy group show an increase in NHE activity and turnover number under conditions mimicking hyperglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of mdm2 gene in x-ray transformed mouse 10t1/2 cell clones.

Radiation-induced malignant transformation develops by a stepwise accumulation of molecular changes including mutation, amplification or overexpression of certain genes. Amplification and/or overexpression of mdm2 may be one of several molecular mechanisms for an altered growth control leading to the transformed phenotype. In the present investigation, we examined amplification, level of expression as well as mutation of mdm2 in radiation-transformed mouse C3H 10T1/2 cell clones. None of the clones examined showed structural changes of the mdm2 gene. However, mdm2 was amplified in 8 of 30 and overexpressed in 3 of 11 independent X-ray (600 cGy) transformed 10T1/2 cell clones, as compared with nontransformed, control or ultraviolet light (UVL)-transformed clones. None of the clones showing amplification and overexpression of mdm2 were among the 9 with alterations in the p53 gene. These results suggest that although amplification of the mdm2 gene may play a role in the transformation of some 10T1/2 cells, radiation-induced malignant transformation probably arises as a consequence of genetic events that involve several different pathways.

Abnormal Na+/H+ antiporter phenotype and turnover of immortalized lymphoblasts from type 1 diabetic patients with nephropathy.

Cellular Na+/H+ exchanger (NHE) activity is elevated in type 1 diabetic patients with nephropathy and patients with essential hypertension. The characteristics of this NHE phenotype in hypertension (raised Vmax and a lowered Hill coefficient) are preserved in Epstein-Barr virus-transformed lymphoblasts from hypertensive patients. In this study, we have determined NHE kinetics in cultured lymphoblasts from diabetic patients with and without nephropathy, with nondiabetic controls, using fluorometry with the pH indicator 2,7'-bis-(carboxyethyl)-5,6-carboxyfluorescein and estimation of NHE isoform 1 (NHE-1) density with specific polyclonal antibodies. The Vmax of NHE was elevated significantly, and the Hill coefficient for internal H+ binding was lowered in cells from patients with diabetic nephropathy compared with both normal controls and normoalbuminuric diabetic patients. NHE-1 density as measured by Western blotting was similar in all groups. The turnover number of NHE-1 was thus elevated in cells from nephropathy patients. This phenotype in cells from diabetic nephropathy patients resembles that in essential hypertension and suggests that such patients may have a predisposition to hypertension. Moreover, as these changes persist in cultured lymphoblasts in vitro, these cells should provide a cell culture model to further define the basic mechanisms leading to NHE activation in diabetic nephropathy.

Search for oncogene mutations in X-ray-transformed mouse 10T1/2 cells by denaturing gradient gel electrophoresis blotting.

We have sought evidence for possible mutations within the c-myc, c-Ha-ras and c-Ki-ras loci of X-ray-transformed mouse C3H10T1/2 cell clones using the denaturing gradient gel electrophoresis (DGGE) blot technique. This highly sensitive method was developed to detect any mutations (e.g. single base changes, small deletions) in genomic DNA, by measuring differences in the melting behaviour of short DNA fragments (50-800 bp) obtained by digestion of genomic DNA with several specific 4 bp recognition site restriction enzymes. In this study, genomic DNAs derived from 23 X-ray-transformed clones were digested with several restriction enzymes, electrophorezed on denaturing gradient gel and hybridized to c-myc, c-Ha-ras and c-Ki-ras cDNA probes. No alterations in melting patterns were observed for any of these oncogenes as compared with DNA from 18 control, non-irradiated wild-type 10T1/2 cell clones, suggesting that transformation was not associated with mutation of these genes nor with changes in their patterns of methylation. However, our screening of the large portion of exons 2 and 3 of c-myc as well as of exons 1 and 2 of c-Ha-ras gene cannot exclude the possibility that some sequence differences in the high melting domains of examined fragments were not detected by this assay.

Application of denaturing gradient gel blots to detect p53 mutations in X-ray-transformed mouse C3H 10T1/2 clones.

The denaturing-gradient gel electrophoresis (DGGE) blot technique is a highly sensitive method developed to detect sequence differences (e.g., single base changes and small deletions) in genomic DNA. We used this method to screen for mutations within the p53 locus of independent X-ray-transformed C3H 10T1/2 cell clones. Genomic DNA from 29 transformed clones was digested with Haell and Hinfl endonucleases, electrophoresed on denaturing gradient gels, electroblotted onto nylon membranes, and hybridized to a radioactive p53 cDNA probe. Changes in the melting pattern of p53 were observed in DNAs from 9 of 29 X-ray-transformed clones examined. No changes were found in untransformed, wild-type 10T1/2 cell clones. These results indicate that X-ray-induced p53 mutations may contribute to cell transformation.

High-frequency DNA sequence polymorphisms in the insulin receptor gene detected by denaturing gradient gel blots.

A limiting factor in the study of genetic determinants of human disorders is the availability of informative DNA markers. In this report, we describe an application of the denaturing gradient gel blot method for detecting high-frequency DNA sequence polymorphisms in the human insulin receptor locus. Using two restriction enzymes and cDNA probes for the insulin receptor, we found five DNA polymorphisms. The probe that contained exons 4-10 of the insulin receptor gene detected two two-allelic polymorphisms in HinfI digests, one at denaturant concentrations of 38%/39% and the other at 46%/48%. The probe that contained exons 14-22 detected three two-allelic polymorphisms in Sau96I digests, the first at denaturant concentrations of 34%/35%, the second at 38%/39%, and the third at 46%/47%. All these DNA polymorphisms segregated in families in a Mendelian fashion, and the allelic distribution for each of them did not deviate from Hardy-Weinberg equilibrium. The identified polymorphisms were in linkage equilibrium and provided sufficient genetic information to determine parental haplotypes at the insulin receptor locus in small two-generation families. The denaturing gradient gel blot method is a very sensitive technique for identifying sequence polymorphisms in genomic DNA; its application will facilitate the search for genes involved in the development of many inherited disorders.

Molecular analysis of DNA isolated from the different stages of x-ray-induced transformation in vitro.

A major challenge in radiation carcinogenesis is to identify the cellular gene or genes involved in initiating the process. We examined the transforming activities of DNAs obtained from C3H10T1/2 cells during x-ray-induced morphological transformation. DNAs extracted from mass cultures of 10T1/2 cells at different times after irradiation with 600 rad and from type III-transformed foci were transfected into NIH 3T3 cells. The results indicate that certain oncogenes are activated beginning 3 wk after irradiation, well before the appearance of macroscopically visible transformed foci. For DNA isolated from x-ray-transformed 10T1/2 cells (type III foci), the frequencies of transfection were 0.003-0.11 foci/microgram of genomic DNA with NIH 3T3 cells and 0.004-0.04 foci/microgram genomic DNA using 10T1/2 cells as recipients. Southern blot analysis of DNAs obtained from 23 primary transfectants and from 23 x-ray-transformed cell lines indicated no gross rearrangements or amplification of any of the 14 oncogenes screened (v-Ha-ras, v-Ki-ras, N-ras, v-myc, v-raf, v-src, v-fes, v-abl, v-mos, v-erbA, v-erbB, v-myb, v-fos, v-sis). This suggests that x-irradiation may activate as yet unidentified oncogenes. The occurrence of positive transfection 3 wk after irradiation is discussed in terms of the hypothesis that transformation may not occur as a direct consequence of the exposure to x-rays but develops as a rare event in the progeny of the irradiated cells at some later time, as a consequence of the delayed activation of certain genes.

Effect of duration of exposure to benzo(a)pyrene diol-epoxide on neoplastic transformation, mutagenesis, cytotoxicity, and total covalent binding to DNA of rodent cells.

We examined the effect of different durations of exposure (20 sec to 24 hr) to (+/-) 7-beta,8 alpha-dihydroxy-9 alpha, 10 alpha -epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE I) on the induction of transformation in C3H/10T 1/2 cells and of mutations to 6-thioguanine resistance in Chinese hamster ovary cells (CHO), as well as on BPDE I-DNA binding in these two cell lines. A 20-sec exposure of the cells to BPDE I was sufficient to induce mutations and morphological transformation in vitro. However, the transformation frequency in CH3 mouse-embryo-derived 10T 1/2 cells increased twofold and the frequency of mutations in CHO cells sixfold when the exposure time to BPDE I was increased from 20 sec to 8 h. Cytotoxicity increased under similar conditions. A large number of BPDE I-DNA adducts were formed in both cell lines within the first 15-min of exposure of the cells to this ultimate carcinogen. The total covalent binding did not increase with longer than 15-min incubation times. These results suggest that in addition to its covalent binding to DNA, BPDE I may influence other cellular mechanism(s) that are responsible for the initiation of transformation and mutagenesis.

Effect of aliphatic amides on oncogenic transformation, sister chromatid exchanges, and mutations induced by cyclopenta[cd]-pyrene and benzo[a]pyrene.

We examined the effects of the aliphatic amides isopropyl-valeramide (IVA) and allylisopropylacetamide (AIA) on oncogenic transformation and sister chromatid exchanges (SCE) induced by cyclopenta[cd]pyrene (CPP) and benzo[a]pyrene (B[a]P) in C3H/10T1/2 cells and on B[a]Pdiol-epoxide (BPDE)-induced mutation at the HGPRT locus in Chinese hamster ovary (CHO) cells. IVA and AIA significantly suppressed B[a]P and CPP transformation in vitro. Both amides were effective when given just prior to, simultaneously with, or 24 h after carcinogen exposure. On the other hand, IVA and AIA did not affect cytotoxicity, the frequencies of SCE induced by CPP or B[a]P, nor BPDE-induced mutations in CHO cells. These and previous results suggest that the mechanism of inhibition of transformation by IVA or AIA may be very specific and probably not related to the early initiation event in oncogenic transformation in vitro.

Influence of isopropylvaleramide and allylisopropylacetamide on transformation of C3H/10T1/2 cells induced by benzo[a]pyrene derivatives.

We examined the effect of aliphatic amides isopropylvaleramide (IVA) and allylisopropylacetamide (AIA) on the oncogenic transformation of C3H/10T1/2 cells induced by benzo[a]pyrene (B[a]P) or its proximate and ultimate metabolites (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (B[a]P-diol-epoxide), respectively. IVA and AIA given prior to, simultaneously with, or for 24 h intervals beginning up to 48 h after removal of carcinogens significantly suppressed transformation induced by B[a]P or the 7,8-diol metabolite. Both modifiers were most effective when added for 24 h immediately following carcinogen exposure. IVA and AIA were also very potent inhibitors of B[a]P-diol-epoxide transformation; however they were most effective when added for 24 h simultaneously with the B[a]P-diol-epoxide. No significant difference in B[a]P-diol-epoxide binding to DNA in C3H/10T1/2 cells was observed during 1 or 24 h exposure to this carcinogen in the presence or absence of IVA or AIA. Neither modifier affected X-ray transformation when added for 24 h immediately following X-irradiation of C3H/10T1/2 cells. These results suggest that AIA and especially IVA might be important tools in studies directed at non-metabolic aspects of B[a]P carcinogenesis.