Cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) inhibits growth of a broad spectrum of cancer cells derived from solid tumors.

In an effort to investigate the role of creatine kinase and its substrates in malignancy we have tested the effect of cyclocreatine [1-carboxymethyl-2-iminoimidazolidine (CCr)] on the growth of tumor cells in vitro and in vivo. CCr is phosphorylated by creatine kinase to yield a synthetic phosphagen [(N-phosphorylcyclocreatine (CCr approximately P)] with thermodynamic and kinetic properties distinct from those of creatine phosphate. We show that CCr accumulates as CCr approximately P in tumor cells expressing a high level of creatine kinase, and that the accumulation of this phosphagen is detrimental to tumor cell growth. Tumor cell lines expressing a low level of creatine kinase accumulate much less CCr approximately P, and consequently are growth inhibited only at higher concentrations of CCr. When these resistant cells are transfected with a creatine kinase B expression vector, they express creatine kinase, accumulate CCr approximately P, and are growth inhibited. In vivo, in nude mouse xenografts, the rate of growth of a high creatine kinase expressing tumor cell line is inhibited in animals fed 1% CCr. Our results indicate that CCr inhibits the growth of tumor cells in vitro and in vivo.

Evidence for interaction of different eukaryotic transcriptional activators with distinct cellular targets.

The adenovirus E1a protein (E1a), a potent transcription activator, contains a transcriptional activating region. Compared with previously described cellular and viral activators, E1a's activating region has unusual structural properties. It seems that E1a's activating region interacts with a cellular target not required for the function of transcriptional activators with 'acidic' activating regions. By contrast, the target of an acidic activating region is required both by acidic activators and by E1a. It is proposed that the cellular target of E1a's activating region is an 'adaptor' that allows E1a to interact with the basic transcriptional machinery.

Induction of a cellular enzyme for energy metabolism by transforming domains of adenovirus E1a.

Brain creatine kinase is a major enzyme of cellular energy metabolism. It is overexpressed in a wide range of tumor cell lines and is used as a tumor marker. We reported recently that the promoter of the human gene has a strong sequence similarity to the adenovirus E2E promoter. This similarity suggested that the brain creatine kinase gene may be regulated by the viral activator E1a. Experiments reported here showed that both enzyme activity and mRNA levels were induced by the oncogenic products of the E1a region of adenovirus type 5, but unlike the viral E2E promoter, which is induced predominantly by E1a domain 3, brain creatine kinase induction required domains 1 and 2. These domains are important for transformation and for the association of E1a with the retinoblastoma gene product and other cellular proteins. The induction by an oncogene of a cellular gene for energy metabolism may be of significance for the metabolic events that take place after oncogenic activation.

Transcription activation by the adenovirus E1a protein.

The adenovirus E1a protein stimulates transcription of a wide variety of viral and cellular genes. It is shown here that E1a has the two functions characteristic of a typical cellular activator: one direct E1a to the promoter, perhaps by interacting with a DNA-bound protein, and the other, an activating region, enables the bound activator to stimulate transcription.

Functional domains of adenovirus type 5 E1a proteins.

Adenovirus E1a proteins function in transcriptional activation, transcriptional repression, cellular DNA synthesis induction, and cellular transformation. Here we examine the role of the previously undefined E1a region 1, the last of three conserved E1a regions to be characterized. Region 1 is required for transcriptional repression, transformation, and DNA synthesis induction, but not transcriptional activation. These results support our previous suggestion that transcriptional repression is the basis of E1a-mediated transformation. Two conserved regions (regions 1 and 2), present in both early E1a proteins, are essential for transcriptional repression, transformation, and induction of DNA synthesis. In contrast, mutagenesis suggests that transcriptional activation requires only 49 amino acids (region 3) unique to the 289 amino acid E1a protein. This we prove by demonstrating that a 49 amino acid region 3 synthetic peptide efficiently activates an E1a-inducible promoter. This peptide is the smallest known protein fragment functioning as a transcriptional activator.

Structure and expression of HLA-DQ alpha and -DX alpha genes: interallelic alternate splicing of the HLA-DQ alpha gene and functional splicing of the HLA-DQ alpha gene using a retroviral vector.

The nucleotide sequences of the two closely related HLA-DQ alpha and HLA-DX alpha genes have been determined. Exons coding for the signal peptide, alpha 2 and transmembrane domains are 94-99% homologous, whereas the alpha 1 exon and the promoter region have diverged as much as or more than introns and the 3' untranslated region. The promoter regions of both genes contain two short sequences thought to be important for regulation of transcription by gamma-interferon. Transfection studies established that the DQ alpha and DQ beta genes encode the HLA-DQ antigen. Transcripts of varying length are produced from different alleles as the result of the use of alternate splice and polyadenylation signals at the 3' end of the DQ alpha gene. Thus typing at the DQ alpha locus can be achieved by Northern blot analysis. No transcript of DX alpha was detected in B lymphocytes. The DX alpha gene was accurately spliced when introduced into a retroviral vector, suggesting that the lack of expression of DX alpha is not due to aberrant splice signals.

An adenovirus E1a protein region required for transformation and transcriptional repression.

The adenovirus E1a region encodes two closely related gene products: 243 and 289 amino acid phosphoproteins. These proteins differ in their primary sequence only by 46 amino acids unique to the 289 amino acid protein. By constructing single-base substitution mutants we localized two functional regions of these E1a proteins: one required for efficient transcriptional activation, another required for efficient transcriptional repression. The 289 amino acid protein contains both regions and appears to function primarily as a transcriptional activator. The 243 amino acid protein lacks the transcriptional activation domain and appears to function primarily as a transcriptional repressor. Mutations within a highly conserved region define a novel class of transformation-defective mutants. These mutant E1a proteins can still efficiently activate transcription of early viral and cellular genes but cannot repress transcription of target genes. The fact that viral transformation may require a transcriptional repression function provides new insights into the mechanism by which adenovirus transforms cells.

Genetic mapping of a human class II antigen beta-chain cDNA clone to the SB region of the HLA complex.

A class II antigen beta-chain cDNA clone was isolated from a human B-cell cDNA library by using as a probe the murine I-A beta gene. This cDNA clone, pHA beta, was shown to be distinct from the DC beta- and DR beta-related loci by DNA sequence analysis, thus suggesting that it might correspond to a third polymorphic human class II locus, SB, which encodes secondary B-cell antigens. Genetic mapping of this beta-chain cDNA clone to the SB region was performed by the blot hybridization procedure. We showed that (i) within panels of HLA-DR homozygous human B-cell lines and of unrelated individuals who have been typed for HLA antigens, differential mobility of DNA fragments segregated with distinct SB genotypes; (ii) gamma-ray-induced deletion mutants that have lost the expression of DR or DC/MT antigens but maintain SB expression preserved a pattern consistent with (a) their SB phenotype and (b) the genetic independence of the SB locus with respect to DR and DC/MT; and (iii) within an informative family, two siblings differing only for one allele at the SB locus (because of the occurrence of an internal recombination between DR and GLO) and otherwise HLA identical exhibited a restriction enzyme polymorphism linked to the SB locus. Therefore, all available data are compatible with identity between HA beta and SB beta.