Growth factor regulated expression of poly(A)+ binding protein messenger RNA.

A 72,000 mol wt protein designated PABP binds to the poly(A)+ track of messenger RNAs with high affinity and has been suggested to play an important role in mRNA metabolism in eucaryotic cells. We have employed a human PABP cDNA probe to study the expression of this gene at the mRNA level in BALB/c3T3 mouse cells under different growth conditions and in exponentially growing HeLa cells throughout the cell division cycle. We describe experiments which establish that in BALB/c3T3 cells the expression of this gene is growth factor regulated. Moreover, the gene behaves like a primary response gene in that its induction in quiescent cells does not require the prior synthesis of other growth factor-regulated proteins. In exponentially growing HeLa cells PABP mRNA is expressed throughout the cell division cycle indicating that the expression of this gene is not limited to a specific phase of the cell cycle.

Regulation of the proliferating cell nuclear antigen cyclin and thymidine kinase mRNA levels by growth factors.

The enzymes of the DNA synthesizing machinery constitute a group of gene products that are generally expressed co-ordinately at the G1/S boundary of the cell cycle. We have investigated how growth factors regulate the steady-state mRNA levels of two of these genes, the PCNA (proliferating cell nuclear antigen)/cyclin and the thymidine kinase genes. To detect the PCNA/cyclin mRNA, we isolated a cDNA clone from a human library. Two different cell lines were used for these studies: BALB/c3T3 cells, which are exquisitely sensitive to growth factors, and ts13 cells, a temperature-sensitive (ts) mutant of the cell cycle, which arrests in G1 at the restrictive temperature. The steady-state levels of the RNAs for these two genes under different growth conditions were also compared with the levels of histone H3 RNA which are good indicators of the fraction of cells in S phase. Both PCNA/cyclin and thymidine kinase genes share two fundamental characteristics, i.e. they are not inducible in a G1-specific ts mutant of the cell cycle at the restrictive temperature and their expression is inhibited by cycloheximide, indicating that unlike early growth-regulated genes, they require the previous expression of other growth-regulated genes. However, the two genes also show differences, the most notable being that PCNA/cyclin is inducible by epidermal growth factor alone, while thymidine kinase is not.

Inhibition of cellular proliferation by antisense oligodeoxynucleotides to PCNA cyclin.

The proliferating cell nuclear antigen (PCNA or cyclin) is a nuclear protein recently identified as a cofactor of DNA polymerase delta. When exponentially growing Balb/c3T3 cells are exposed to antisense oligodeoxynucleotides to PCNA, both DNA synthesis and mitosis are completely suppressed. A corresponding sense oligodeoxynucleotide has no inhibitory effects. These experiments indicate that PCNA (cyclin) is important in cellular DNA synthesis and in cell cycle progression.

Role of the promoter in the regulation of the thymidine kinase gene.

To identify the regulatory elements of the human thymidine kinase (TK) gene, we have established stable cell lines carrying different chimeric constructs of the TK gene. Our results can be summarized as follows. (i) When the TK coding sequence is under the control of the calcyclin promoter (a promoter that is activated when G0 cells are stimulated by growth factors), TK mRNA levels are higher in G1-arrested cells than in proliferating cells; (ii) when the TK coding sequence is under the control of the promoter of heat shock protein HSP70, steady-state levels of TK mRNA are highest after heat shock, regardless of the position of the cells in the cell cycle; (iii) the bacterial CAT gene under the control of the human TK promoter is maximally expressed in the S phase; (iv) the TK cDNA driven by the simian virus 40 promoter is also maximally expressed in the S phase; and (v) TK enzyme activity is always at a maximum in the S phase, even when the levels of TK mRNA are highest in nonproliferating cells. We conclude that although the TK coding sequence may also play some role, the TK promoter has an important role in the cell cycle regulation of TK mRNA levels.

Gene transcription in a temperature-sensitive mutant of RNA polymerase II.

We have studied the transcription rates and the steady-state levels of cytoplasmic mRNA of selected genes in tsAF8 cells, a temperature-sensitive mutant of RNA polymerase II. At the restrictive temperature, the transcription rates decrease uniformly for all genes examined, while the corresponding decreases in steady-state levels of cytoplasmic mRNA vary for individual genes. tsAF8 cells can be advantageously used to study the half-line of mRNA without the use of perturbing drugs.

Stimulation of cellular DNA synthesis by wild type and mutant bovine papillomavirus DNA.

Microinjection of recombinant plasmids containing bovine papillomavirus type 1 DNA into the nuclei of mouse C127 cells results in the stimulation of cellular DNA synthesis. Mutations in the viral E2 gene have no apparent effect on this activity even though the same mutations prevent efficient C127 cell focus formation and inhibit transactivation by this gene.

Omental lymphoid organ as a source of macrophage colony stimulating activity in peritoneal cavity.

To test the hypothesis that the omental lymphoid organ (OLO) made by peritoneal milky spots is either a source of haemopoietic (macrophage) progenitors or growth factors we attempted to culture OLO cells in vitro in a variety of assay combinations. With the culture in vitro in semisolid agar it was found that OLO cells do not form granulocyte-macrophage or macrophage colonies in response to stimulants. However, when in the same assay marrow cells were used as the targets and OLO-related preparations as stimulants it was observed that marrow cells formed exclusively macrophage colonies. These marrow cells, in response to stimulants derived from other organs, produced granulocyte-macrophage, granulocyte and macrophage colonies. OLO-related preparations tested for macrophage-colony stimulating activity included partly purified medium conditioned by OLO cells derived from mice, either injected with endotoxin or not, and medium conditioned by OLO cells after 14 days, liquid culture in vitro. While these results were observed in Swiss mice, C3H/W mice, which are genetically endotoxin-unresponsive, failed to show this reaction. These data may suggest that the local production of macrophage-colony stimulating activity in the peritoneal cavity could be one physiological role for OLO. OLO is the first organ in adult mice identified to stimulate exclusively macrophage colony growth, and not granulocyte-macrophage or pure granulocyte colonies.

Success of bone marrow transplantation in congenital Diamond-Blackfan anaemia: a case report.

A 6-yr-old girl with congenital corticosteroid-resistant pure red cell aplasia was treated with bone marrow transplant from her HLA-identical, MLC-unreactive sister in November 1984 following conditioning with busulfan and cyclophosphamide. Full engraftment was obtained and the patient at 21 months post-transplant is in excellent clinical condition maintaining normal red cell counts. We conclude that BMT should be considered as a therapy for at least the most severe cases of Diamond-Blackfan anaemia resistant to corticosteroids. Successful outcome of this therapy provides an argument for the stem cell origin of this disorder.