Mechanogenetic regulation of transcription.

In many biological systems mechanical forces regulate gene expression: in bacteria changes in turgor pressure cause a deformation of the membrane and induce the expression of osmoregulatory genes; in plants gravity regulates cell growth ('geotropism'); in mammals stretching a muscle induces hypertrophy which is accompanied by qualitative changes in protein synthesis. Consequently, the term 'mechanogenetic control' seems to be a suitable common name for all these processes. The mechanism by which mechanical factors modulate transcriptional activity is still unknown. The purpose of this review is to bring together data from different fields in order to obtain a better understanding of the mechanogenetic control of cell growth.

The number of binding sites: the Klotz graph analysed.

A semilogarithmic plot (bound versus log of free ligand) has been proposed (Klotz, Science 217, 1247, 1982) to replace advantageously the Scatchard plot. We show that this representation has its own serious practical limitations and conclude that there are no "perfect" solutions: data have to be analysed in the light of all possible models using all available representations.

Activation of alpha-fetoprotein synthesis in rat hepatoma cells with reduced sensitivity to dexamethasone.

The Faza 967 'differentiated', dexamethasone (DEX)-sensitive cell line of Reuber rat hepatoma cells does not synthesize detectable amounts of alpha-fetoprotein (AFP), whereas it does produce albumin. AFP production was activated in 'differentiated' variants of Faza 967 cells with reduced glucocorticoid sensitivity upon culture for several months in the presence of high concentrations of dexamethasone. The stability of AFP production differed among the variants, while albumin synthesis did not change, thus indicating that the regulation of these two genes is not co-ordinated. Using molecular hybridization techniques, we found that the AFP message could not be detected in the non-AFP-producing cells, suggesting that the lack of AFP synthesis most probably originates from a transcriptional block of the AFP gene. AFP-producing and non-AFP-producing variants of Faza 967 cells constitute a valuable model system for studying the regulatory mechanisms involved in the activation and inactivation of the gene coding for the oncodevelopmental protein, AFP.

A lethal deletion on mouse chromosome 7 affects regulation of liver-cell-specific functions: posttranscriptional control of serum protein and transcriptional control of aldolase B synthesis.

Steady-state levels of mRNAs were determined for the serum proteins albumin, alpha-fetoprotein (AFP), and transferrin, as well as for aldolase B in livers of newborn mice homozygous for a radiation-induced lethal deletion (c14CoS) in chromosome 7. Deficiencies in synthesis and secretion of the serum proteins as well as in activities of certain liver-specific enzymes characterize these homozygotes. The results of RNA dot and gel-blot hybridizations with the respective cloned cDNA probes showed a decrease to one-fourth of aldolase B mRNA levels in homozygous mutant livers compared to normal littermates, in contrast to normal levels of mRNA sequences for the three serum proteins in the mutants. Furthermore, the mRNA sequences were shown to be present as mature mRNA molecules in both mutant and normal littermate livers. We suggest that the deficiencies of liver-specific serum proteins and those of the enzymes caused by the lethal deletions around the albino locus on chromosome 7 of the mouse are due to different causes. In the case of the liver-specific enzyme examined here--i.e., aldolase B--control at the level of transcription or of message stability is affected in the homozygous deletion mutants, whereas the deficiencies of serum proteins are not reflected on the mRNA level and owe their origin to an effect on a posttranscriptional or translational level. These results lend further support to the assumption that the deleted portion of the genome includes genes concerned with the control and regulation of liver cell differentiation.

Structural basis for restriction-site polymorphism at the albumin locus in inbred strains of rats.

Two types of variant EcoRI restriction enzyme patterns of albumin-gene DNA fragments are found in different inbred strains of rats and reflect allelic polymorphism. The structural basis of the two allelic forms has been analyzed by mapping the EcoRI fragments using cloned albumin cDNA probes corresponding to the 5' or 3' end of the rat albumin mRNA and different genomic subclones. Additional restriction fragment length polymorphism has been detected using the restriction endonucleases HindIII and MspI. The results suggest that the two allelic variants differ from each other by multiple cleavage-site variations (base-pair substitutions) and by an insertion or deletion of DNA sequences. An extensive DNA sequence variation appears to exist between the two forms of the albumin gene; we have estimated that as much as 4% of the nucleotides in this region varied between the two alleles. All of this genetic variation is found in the intervening sequences of the gene and has no phenotypic manifestation.

Differential DNase I sensitivity of the albumin and alpha-fetoprotein genes in chromatin from rat tissues and cell lines.

We have examined the DNase I sensitivity of the albumin and alpha-fetoprotein (AFP) genes in different rat tissues (adult liver and kidney) and cloned cell lines (hepatoma 7777-C8, JF1 fibroblasts), which show drastic differences in the level of expression of these two genes. This was done by studying the disappearance of defined restriction endonuclease fragments of these genes as a function of limited DNase I digestion. The sensitivity of these genes was compared to that of a gene not expressed in the hepatic cells and to that of a ubiquitously expressed gene. In nuclei from adult rat liver the albumin and AFP genes were preferentially degraded by the nucleolytic action of DNase I, whereas they were not in rat kidney nuclei. In the hepatoma cells the AFP gene was much more sensitive to DNase I digestion than the albumin gene; both genes were very resistant to DNase I action in fibroblastic nuclei. When analyzed in relation to the level of gene expression our results indicate that alterations in the chromatin structure of the albumin and AFP genes might be involved in the early establishment of the tissue-specific potential of overt gene expression; such alterations reflected in an altered DNase I sensitivity do not appear to be responsible for the changes in gene activity occurring during the terminal differentiation of the hepatocyte; and modifications in the chromatin structure of these genes might occur during oncogenic events; these structural modifications could be related to the changes in gene expression observed in hepatocarcinogenic processes.

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat.

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.

Expression of the albumin gene in rat hepatoma cells and their dedifferentiated variants.

Rat hepatoma clones whose cells do and do not produce albumin, as well as somatic hybrid between the two types of cells, have been examined for albumin mRNA. A direct proportionality between the rate of albumin production and the concentration of albumin mRNA sequences was found for all albumin-producing hepatoma and hybrid clones, indicating that rate of synthesis of the protein is determined by the concentration of its mRNA. Albumin-negative dedifferentiated variant and somatic hybrid cells contain fewer than one to five molecules of albumin mRNA per cell; the block in expression of the gene appears to be at the same (probably transcriptional) level in variants and their somatic hybrids.

The endometrial nuclear estradiol receptor of the pregnant cow has a molecular weight of 53,000 in 6 M guanidine-HCl.

Cell nuclei isolated from the endometrium of the 3 months pregnant cow were dialysed against 6 M guanidine-HCl + 0.1 M 2-mercaptoethanol. The resulting material (after eliminating the bulk of DNA by centrifugation) was filtered on a Sepharose 6/b column equilibrated in the above denaturing solvent. The molecular weight of the denatured "nuclear" estradiol receptor, estimated by a method described previously (T. Erdos and J. Fries (1974) Biochem. Biophys. Res. Commun. 58, 932-939), was about 53,000. This value is similar to that of the cytoplasmic receptor (55,000) but dissimilar to that of the cytoplasmic receptor transformed by the action of the Receptor Transforming Factor (35,000) estimated under the same experimental conditions, suggesting that the latter form of the receptor is not the biological precursor of the "nuclear" receptor. However, according to indirect estimates, probably not more than 10% of the "nuclear" receptor has been renatured in these experiments. Therefore the alternative that the results obtained are not representative for the "nuclear" receptor, cannot be excluded.

The effect of passive immunization against estradiol on the regulatory profile and character of labor in the rat.

Treatment of pregnant rats with antiestradiol (A-E2) serum twice a day, starting at 2100 hours on day 19, sharply increased circulating total estradiol (E2) above control values and drastically reduced the biologically active E2 unbound (E2U) to A-E2 in plasma and uterine tissue. This A-E2-induced and--sustained E2U deficiency was not accompanied by similar changes in plasma and tissue progesterone (P), since P decreased similarly in the control and A-E2--treated rats in preparation for parturition. However, in the A-E2 rats the reduction in E2U was accompanied by a small, though significant, decrease in uterine vein prostaglandin F (PGF) during labor. This A-E2--provoked regulatory imbalance significantly altered normal parturition. In comparison with controls, labor in the A-E2 rats was delayed and prolonged by extended intervals between deliveries of individual fetuses of the same litter. The delay in the onset of labor significantly increased the birth weights of the newborn rats. Whether E2U deficiency is directly responsible for the asynchronic myometrial activity that delays and prolongs labor or whether it is mediated by reduced PGF release remains to be determined.

Prevention of the abortifacient action of antiprogesterone serum by progesterone.

Earlier studies1-4 showed that when rats of 10 days' gestation are passively immunized with antiprogesterone (A-P) globulins the biologically available progesterone (P) unbound by A-P (Pu) not only falls precipitously but also remains low for days, despite the rapid clearance of A-P. This finding suggested that the effective reduction of Pu affects P synthesis, probably through action on the fetoplacental unit. If so, pregnancy should be protected from the effect of A-P by P treatment to prevent the reduction of Pu. The present studies demonstrated that if P treatment was delayed for six hours after the administration of A-P, Pu did not return to physiologic levels, and pregnancy did not continue. However, if P was given three hours after A-P, at the same time, or three hours before A-P, the reduction of Pu was short-lived or prevented, and thus pregnancy was protected.

PIP: It has previously been shown that passive immunization of 10-day pregnant rats with antiprogesterone (AP) globulins dramatically reduces the amount of available progesterone (P) unbound (Pu) to AP, even though AP is rapidly removed. In the present study with pregnant rats, it was shown that treatment with P no earlier than 6 hours after administration of AP on Day 10 of pregnancy did not return Pu levels to the normal range, and resulted in termination of pregnancy. However, if P was administered within 3 hours before or after administration of AP, the loss of Pu was transitory or prevented, and pregnancy was maintained.

The critical control of progesterone levels and pregnancy by antiprogesterone.

Circulating plasma progesterone (P) has been quantitatively controlled in the rat "model" through highly specific binding by treatment with anti-P (A-P). Knowing the constant, which characterizes the binding of P to A-P in plasma, sequential assays of circulating A-P and A-P bound total P (Pt) revealed the levels of the biologically active unbound P (Pu). The studies showed that at different stages of gestation the mechanisms through which A-P reduces Pu and terminates pregnancy are the same. However, the doses of AP which effectively reduce Pu and also the critical levels of Pu at which pregnancy terminates are different. The moderate and transient physiologic P-withdrawal (Pw) at midterm permits the continuation of normal gestation, but pregnancy is terminated by a drastic and sustained reduction in Pu. In contrast, when Pu is only slightly and briefly reduced below physiologic levels, pregnancy continues and only retarded conceptus growth signals that Pw occurred. Apparently Pw has to be controlled and measured with "razor's-edge" precision to fully expose and define the regulatory significance of this steroid in the maintenance and termination of pregnancy. Short of this precision, the key regulator of the pregnant uterus will remain buried, as it has been during 40 years, in controversial findings.

A computer simulation of the effect of injected anti-progesterone on progesterone levels of pregnant rats.

Antibodies to progesterone injected into pregnant rats, provoke abortion if the concentration of the biologically active progesterone is reduced to a critical level. By stimulating the biological experiments in the computer we describe the time dependent changes of this progesterone fraction, as a function of the initial concentration of progesterone and of the anti-progesterone injected. Results obtained can be used to aid interpreting the findings of the biological studies and to calculate the concentration of anti-progesterone required to provoke abortion.

The parameters of the binding of estradiol and progesterone to their antibodies in presence of rat plasma.

The binding of estradiol and progesterone to the corresponding antibodies has been studied in system containing : a) antibody and hormone or b) antibody, hormone and rat plasma. In sytem a) the apparent association constant and the concentration of the antibody have been determined and the optimal conditions of a standard antibody-assay have been elaborated. In system b) the specific binding of hormone to antibody in presence of rat plasma has been measured, and the value of the "Operational Association Constant", Kop, has been calculated. Kop has the dimensions of an apparent association constant : it defines the ratio hormone bound/unbound to antibody, as a function of hormone concentration in system b). Based upon these experiments, the extent of hormone-binding to antibody injected to rats can be predicted. When antibody and hormone concentrations are measured in the plasma, the concentrations of hormone bound to antibody can be calculated.

The biological effects of injected antibodies to estradiol-17 beta and to progesterone in pregnant rats.

Estradiol-17 beta (E2) and progesterone (P) levels have been effectively reduced in pregnant rats by injecting them with antibodies which bind E2 or P (A-E2- or A-P) with high specificity. Before, and at daily intervals after treatment with A-E2 or A-P, blood was collected sequentially from the tail vein and the levels of circulating A-E2, A-P total E2 (E2t) and total P (Pt) determined. Having established the relationship between A-E2, E2t, bound E2 (E2b) and unbound E2 (E2u), as well as between A-P, Pt, bound P (Pb) and unbound P (Pu), the concentrations of E2u and Pu could be calculated reliably. Treatment of gestating rats with A-E2 and A-P lowered E2u and Pu levels significantly (P less than 0.001). In comparison with the 14 controls, the 17 A-E2 treated (and thus E2u-deficient) animals showed significant increases in placental weight (P less than 0.001). This effect of A-E2 was readily prevented by replacement therapy with diethylstilbestrol (DES), since A-E2 does not bind DES. In 21 pregnant rats, treatment at day 10 of gestation with only 2 ml A-P (total "Binding Capacity" = 6 mug P) critically lowered the Pu levels and provoked abortion. In contrast, the same treatment in 12 rats at day 6 of gestation (when the P levels are higher than at day 10) failed to provoke the appropriate degree of P-withdrawal (Pw) and abortion, illustrating the quantitative nature of the relationship between effective A-P treatment, Pw and the termination of pregnancy.