Therapeutic targeting of the p53 tumor suppressor gene.

The p53 tumor suppressor gene is a logical target for cancer therapy. Several therapeutic strategies can be envisioned based upon recent advances concerning structure and function of the p53 protein, its interaction with cellular and viral proteins and its roles in repairing DNA, regulating cell division and promoting apoptosis.

Discovery and characterization of an HIV-1 Tat antagonist.

Ro 5-3335, 7-chloro-5-(2-pyrryl)-3H-1,4-benzo-diazepin-2-(H)-one, has been shown to inhibit gene expression controlled by the human immunodeficiency virus-1 (HIV-1) LTR promoter. The inhibition was specific for the viral transcriptional transactivator Tat. The compound did not inhibit the basal activity of the HIV-1 LTR or the activity of promoters not responsive to Tat. Consistent with its mode of action, Ro 5-3335 inhibited HIV-1 replication (IC50 = 0.1-1 microM) by reducing viral RNA synthesis in acutely, as well as chronically, infected cells in vitro. The compound was active against HIV-1 and HIV-2, and AZT-resistant clinical isolates.

Inhibition of HIV replication in acute and chronic infections in vitro by a Tat antagonist.

The human immunodeficiency virus-1 (HIV-1) trans-activator Tat is an attractive target for the development of antiviral drugs because inhibition of Tat would arrest the virus at an early stage. The drug Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepine-2(H)-one], inhibited gene expression by HIV-1 at the level of transcriptional trans-activation by Tat. The compound did not inhibit the basal activity of the promoter. Both Tat and its target sequence TAR were required for the observed inhibitory activity. Ro 5-3335 reduced the amount of cell-associated viral RNA and antigen in acutely, as well as in chronically infected cells in vitro (median inhibition concentration 0.1 to 1 micromolar). Effective inhibition of viral replication was also observed 24 hours after cells were transfected with infectious recombinant HIV-1 DNA. The compound was active against both HIV-1 and HIV-2 and against 3'-azido-3'-deoxythymidine (AZT)-resistant clinical isolates.

Changes in gene expression following exposure of nulli-SCCl murine embryonal carcinoma cells to inducers of differentiation: characterization of a down-regulated mRNA.

cDNA libraries have been generated from Nulli-SCCl murine embryonal carcinoma (EC) cells untreated or treated for 24 h with all-trans retinoic acid (RA) or hexamethylenebisacetamide (HMBA), two chemically unrelated inducers of differentiation of EC cells. The libraries were screened for gene sequences whose expression was differentially regulated by one or both compounds. Of 20,000 cDNA clones screened, only 12 showed reproducible quantitative differences. One of the latter clones (pH 34) has been studied in detail. pH 34 cDNA hybridizes with a polyadenylated RNA (650 nucleotides) which is abundant in untreated Nulli-SCCl EC cells but whose steady-state levels decrease within 6 h of exposure to HMBA, reaching a minimum at 24 h. RA has a less-marked effect on this mRNA. Addition of inducers to the cells in fresh medium produces an early (15 min) transient increase in pH 34 mRNA levels. Nuclear run-on experiments are consistent with the view that the decrease in pH 34 mRNA is due to post-transcriptional events. Subclones of pH 34 in pGEM-4 were used to synthesize mRNA which could be translated in vitro into a 14-kDa protein. DNA sequencing of the pH 34 cDNA revealed that it is 607 bp in length with a single open reading frame capable of encoding a protein of 118 amino acids. Primer extension experiments revealed that the insert contains the full 5' sequence. Comparison with known sequences failed to reveal significant homology with previously sequenced proteins.

Expression of nuclear retinoic acid receptors in wild-type and mutant embryonal carcinoma PCC4.aza1R cells.

Retinoic acid (RA) induces differentiation of murine embryonal carcinoma PCC4.aza1R cells. In this study, the expression of nuclear retinoic acid receptors (RARs) in PCC4.aza1R cells is examined. Analyses of [3H]RA-labeled nuclear extracts prepared from PCC4.aza1R cells by size-exclusion high-performance liquid chromatography demonstrated the presence of a specific RA-binding activity that migrated with a molecular weight of approximately 50,000. More than 95% of this binding activity was associated with the nuclear fraction. In contrast to cytosolic retinoic acid-binding protein, the RARs bound RA analogues of the Ch-series very effectively. Northern blot analyses of total RNA with complementary DNA probes specific for RAR alpha, RAR beta, and RAR gamma showed that PCC4.aza1R cells contain predominantly transcripts encoding RAR alpha and RAR gamma; RAR beta transcripts were undetectable. Treatment of PCC4.aza1R cells with RA increased the levels of RAR beta mRNA in a dose- and time-dependent manner. The RA concentration for half-maximum induction of RAR beta mRNA was 1 nM. An increase in RAR beta mRNA was detectable as early as 2 h after the addition of RA. This increase was not abrogated by cycloheximide, suggesting that protein synthesis is not required for this response. The ability of several retinoids to increase RAR beta mRNA levels in PCC4.aza1R cells correlated well with their binding affinity to the RARs but not with their binding affinity to cytosolic retinoic acid-binding protein. Two mutant cell lines, PCC4(RA)-1 and (RA)-2, which do not undergo differentiation after RA treatment, contained levels of RAR-binding activity very similar to those of the parental cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Incidence and clinical significance of neutralizing antibodies in patients receiving recombinant interferon-alpha 2a.

In clinical trials with a range of doses of recombinant interferon-alpha 2a, we observed a 25% overall incidence of neutralizing antibody development. Careful data analysis did not reveal a relationship between antibody development and therapeutic response. Of 752 patients evaluable for antibody development and clinical response, 31% of the antibody-positive patients and 28% of the antibody-negative patients had therapeutic responses. Although the formation of anti-IFN antibodies seems to be associated with most IFN preparations, incidence and clinical significance have been difficult to determine for a number of reasons. Different assays with differing sensitivities for the detection of IFN antibodies have been utilized in different studies. Additionally, important clinical variables that affect antibody development have often not been carefully controlled or analyzed. The relationship between antibody development and multiple related factors, such as route of administration, dose regimen, cumulative dose, duration of treatment, and the underlying disease, requires clarification. Initial analyses suggest that the median time to antibody development may vary with the dose regime and cumulative dose, as well as the route of administration. Carefully designed prospective studies controlling for influential clinical variables and utilizing standardized assay techniques are required for a meaningful analysis of the antigenic potential of all therapeutic protein products.

Identification and characterization of nuclear retinoic acid-binding activity in human myeloblastic leukemia HL-60 cells.

Specific [3H]retinoic acid (RA)-binding sites in nuclear and cytosolic extracts prepared from human myeloblastic leukemia HL-60 cells have been detected by sucrose density gradient sedimentation and size-exclusion high-performance liquid chromatography (HPLC) analyses. This RA-binding activity migrated as a single peak with an apparent molecular weight of 50,000 and greater than 95% of the total binding activity was associated with the nuclear extract. Nuclear extracts prepared from COS-1 cells transfected with an expression vector for the nuclear RA receptors RAR alpha or RAR beta were enriched (20- to 100-fold) with a RA-binding activity that coeluted by size-exclusion HPLC with the putative RAR from HL-60 cells. The HL-60 nuclear receptor exhibited high-affinity binding of RA and its benzoic acid analogs Ch55, Ch30, Ro 13-7410, and SRI 6409-40 and low-affinity binding of retinol, Ro 8-8717, and SRI 5442-60, correlating well with the biological activity of these compounds in HL-60 cells. Saturation binding and Scatchard plot analyses of the binding of RA to the nuclear HL-60 receptor yielded an apparent dissociation constant of approximately 0.46 nM and 1400 +/- 100 receptor sites per cell. Northern blot analyses of poly(A)+ RNA with cDNA probes specific for RAR alpha and RAR beta indicated that HL-60 cells contain predominantly transcripts encoded by the RAR alpha gene. Our results suggest that the observed nuclear RA-binding activity in HL-60 cells might mediate the action of RA in these cells.

Regulation and patterns of endogenous and exogenous gene expression during differentiation of embryonal carcinoma cells.

Embryonal carcinoma (EC) cells offer an interesting model system for evaluating differentiation because the cells are pluripotent, thus resembling germ cells and embryonic stem cells, and because a number of agents have been defined that are capable of promoting the differentiation of these cells. This chapter examines how EC cells might be triggered to differentiate, with emphasis on retinoic acid because this compound is a potent, naturally occurring inducer that has been studied extensively in this system. The nature of alterations in gene expression during EC cell differentiation is reviewed from the perspective of evaluating whether these changes are likely to be responsible for, or a result of, the differentiation event. Finally, we consider in molecular terms why EC cells, but not their differentiated derivatives, are refractory to the expression of many viral genomes following infection. Based upon these studies, we propose that fundamental changes in gene expression that are observed when differentiation is triggered in EC cells are likely to be due to the disappearance or neutralization of strong repressor elements.

The therapeutic activity in cancer of IL-2 in combination with other cytokines.

Animal studies have been carried out to assess the antitumour efficacy of recombinant interleukin-2 (rIL-2) in combination with other cytokines. In several murine tumour models, rIL-2 in combination with recombinant alpha interferon (rIFN-alpha) elicits a potent antitumour response which is often greater than that which can be reached with the individual agents at non-toxic doses. By contrast, recombinant gamma interferon (rIFN-gamma) usually fails to potentiate the antitumour response to rIL-2. Recombinant alpha tumour necrosis factor (rTNF) can synergize with rIL-2 in some circumstances, but, as with the rIL-2/rIFN-alpha combination, the correct regimen is critical for generating a potent response without overt toxicity. Although appropriate cytokine combinations can lead to markedly enhanced tumour infiltration by lymphocytes, it is not clear that only a single type of lymphocyte is invariably involved in the antitumour response or, for that matter, the toxic side effects; nor has the mechanism of action of any of the cytokines in the therapeutic action been unequivocally elucidated. Finally, results of early clinical studies appear to be consistent with results in preclinical models: promising clinical responses to the combination of rIL-2 and rIFN-alpha have already been observed and further study is merited.

Effects of dietary retinoids upon growth and differentiation of tumors derived from several murine embryonal carcinoma cell lines.

We have examined the effects of dietary retinoids upon the growth and differentiation of seven embryonal carcinoma lines in mice. The control diet contained 4000 IU/mg retinyl palmitate; the other diets contained 2 x 10(5) IU/mg retinyl palmitate, 50 mg/kg all-trans-retinoic acid (RA), 100 mg/kg RA, and no retinoid. The RA-containing diets had little influence on tumor latency or incidence but did suppress growth of many of the tumors. Decreased tumor mass was, in most but not all instances, accompanied by an increased proportion of differentiated cells. Increased differentiation was most commonly quantitative rather than qualitative; i.e., there was a larger proportion of the same types of differentiated cells seen in tumors from the control diet group rather than an increase in the spectrum of cell types observed. Notably, tumors from two differentiation-defective embryonal carcinoma lines were refractory to both the differentiation-inducing and growth-suppressing properties of dietary RA. Taken together, our results suggest that dietary RA can reduce teratocarcinoma growth in part by promoting differentiation but that other mechanisms are likely to be involved. The therapeutic benefits that we observed with dietary RA were compromised by adverse effects, including failure of the mice to gain weight as effectively as those on the control diet. The effects of elevated levels of retinyl palmitate, or its omission from the diet, were much less striking than that of RA. Both modifications tended to decrease tumor latency but had little effect, if any, upon ultimate tumor mass. Elimination of retinoid from the diet failed to significantly reduce degree of differentiation in tumors which normally differentiate extensively in animals on retinoid-containing diets. Excess retinyl palmitate led to a marginal increase in differentiation in F9 tumors and a statistically significant increase in differentiation in OC15-S1 tumors. Tumors from other embryonal carcinoma lines did not contain elevated levels of differentiated cells. The interpretation of these results is complicated by our observations that although our dietary alteration in levels of palmitate were dramatic, they resulted in much more modest differences in circulating retinoid levels when compared with mice on the control diet.

Differential uptake, binding, and metabolism of retinol and retinoic acid by 10T1/2 cells.

Previous studies have shown that all-trans-retinoic acid fails to inhibit chemically induced transformation in 10T1/2 cells except at toxic levels, whereas retinol and many synthetic retinoids are potent inhibitors. In contrast, in many systems retinoic acid is a more effective modulator of differentiation and carcinogenesis than is retinol. In any attempt to explain this anomaly, we have studied the differential metabolism of retinoic acid and retinol by 10T1/2 cells and by their initiated and transformed derivatives, and have also reexamined these cells for the presence of retinoid-binding proteins. Whereas retinoic acid was depleted from the medium bathing 10T1/2 and initiated 10T1/2 cells within 48 h of treatment, retinol was concentrated 500-fold by these cells, and disappeared from the culture medium no faster than from cell-free medium. Retinoic acid metabolism by a number of transformed cell lines varied widely. There was no apparent correlation between metabolizing ability and transforming agent (methylcholanthrene, X-rays, fission spectrum neutrons, and plasmid oncogene transfection). Uptake of retinoic acid was seen in all cell lines and was not correlated with its metabolism. Retinol was metabolized minimally by all cell lines tested; metabolism of retinol was not correlated with retinoic acid metabolizing ability. Retinoic acid-induced growth inhibition and cytotoxicity were not correlated with metabolizing ability, suggesting that the rate of metabolism of retinoic acid is not a major determinant of its acute biological effects. Using sensitive radioimmunoassays, cellular retinoic acid- (CRABP) and retino-binding proteins (CRBP) were both detected in 10T1/2 and initiated 10T1/2 cells. CRABP levels of about 16 ng/10(6) cells were about 4-fold higher than CRBP levels. Therefore, lack of CRABP does not explain the failure of retinoic acid to inhibit carcinogen-induced transformation in these cells. These studies suggest that the inability of retinoic acid to inhibit transformation in the 10T1/2 cell system may be due to its rapid metabolism and clearance from the medium. On the other hand, the high cellular uptake and stability of retinol in these cells could be an important factor in the inhibition of neoplastic transformation by this retinoid.