A mutation in the promoter of the multidrug resistance gene (MDR1) in human hematological malignancies may contribute to the pathogenesis of resistant disease.

The overexpression of the multidrug resistance gene MDR1 has been found to be associated with therapy-resistance in hematological malignancies. Yet the cellular mechanisms underlying this increased expression are completely unknown. Point mutations in the MDR1 promoter have been found in osteogenic sarcoma (Stein et al., Eur J of Cancer, 30A: 1541-1545, 1994). We therefore analyzed DNA from hematological malignancies for MDR1 promoter point mutations. Two pairs of overlapping PCR primers were designed which did not amplify the MDR3 gene. Amplified DNA was screened using single strand conformation polymorphism (SSCP). 139 patients and 93 normal controls were studied. Fifteen patients (11%) were found to have abnormal bands on the SSCP analysis. Of these, 9 had acute myeloid leukemia (AML), 4 chronic lymphocytic leukemia (CLL), 1 acute lymphocytic leukemia (ALL), and 1 nonHodgkin's lymphoma (NHL). Sequence analysis revealed that all patients were heterozygous for a point mutation in the promoter (T-C transition at +8). Four normals (4%) were found to be heterozygous for the mutation. Confirmation of the mutation was performed by oligonucleotide probe hybridization. All but two of the AML patients have died due to chemoresistant disease (one is lost to followup). Of the CLL patients, one is alive with progressive disease, and the others have died. Further studies will assess the effect of this mutation on MDR1 gene transcription.

Role of Escherichia coli cspA promoter sequences and adaptation of translational apparatus in the cold shock response.

A shift in growth temperature from 37 degrees C to 15 degrees C leads to a dramatic increase in the level of CspA, the major cold shock protein of Escherichia coli. To investigate the molecular basis of this induction, we considered the relevance of transcriptional and posttranscriptional controls by analyzing the steady-state levels of transcripts and the expression of reporter genes in cells carrying a set of cspA promoter fragments of variable length fused to lacZ or cat genes. We demonstrate that: (i) the core cspA promoter (from -40 to +16) responds to cold shock and a mutation at -36 increases the relative activity of the promoter at low temperature by threefold; (ii) the sequences upstream of -40 have a positive effect on expression at 37 degrees C, but no effect on the cold shock response; (iii) by virtue of their influence on mRNA stability, the downstream sequences (from +81 to +165) reduce expression at 37 degrees C and increase the intensity of the cold shock response; (iv) mutations in the GCACATCA and CCAAT motifs, present at +1/-4 and between the -10 and -35 elements, respectively, do not affect the cold shock response of the cspA promoter; (v) following cold shock, a modification of the protein synthetic machinery takes place that allows preferential translation of cspA mRNA relative to the non-cold shock cat and lacZ mRNAs. The quantitatively modest transcriptional activation shown by the core promoter of cspA following cold shock suggests that transcriptional activation can significantly contribute to cold shock induction only when coupled to posttranscriptional controls, such as alterations in mRNA stability and the translational apparatus.

Differential mRNA stability of the cspA gene in the cold-shock response of Escherichia coli.

Exposure of bacterial cells to temperature changes induces the synthesis of a set proteins. We investigated the control of expression of the cspA gene, coding for the major cold-shock protein of Escherichia coli. This protein was shown to be transiently induced upon shift to low temperature. We demonstrated that the cspA mRNA is extremely unstable at 37 degrees C with a half-life of approx. 10 s. Upon shift to 15 degrees C cspA mRNA becomes highly stable. This mRNA stability is transient and is lost once the cells are adapted to the low temperature. Transcription fusions of lacZ containing part or most of the cspA gene do not show the rapid degradation at high temperature. Our results suggest that mRNA stability plays a major role in the control of the cspA gene. The expression of cspA is also regulated, to a smaller extent, by the relative increase in transcription after transfer to low temperature. A model by which cspA mRNA is regulated in response to temperature shift is discussed.

The effects of oxytocin on intracranial pressure.

The commonly used vasodilators sodium nitroprusside and nitroglycerine increase the intracranial pressure (ICP). Oxytocin is also a vasodilator although its primary effect is contraction of uterine smooth muscle. We therefore studied the effect of oxytocin on the ICP of cats. Twelve cats were assigned to two equal groups. One group had normal ICP and in the other the ICP was artificially increased by inflating a balloon placed in the extradural space. All of the cats initially received an oxytocin infusion for 15 min. After a period of stabilization, an additional dose of oxytocin was injected as a bolus. Oxytocin administered as an infusion did not change the ICP significantly in any of the cats. Oxytocin administered as a bolus increased the ICP from 27 +/- 4 to 31 +/- 2 mm Hg in the cats with artificially increased ICP. There were no significant changes in the mean blood pressure or heart rate in either group.

Effect of suxamethonium on the ICP of cats with and without thiopentone pretreatment.

Twelve cats were assigned equally to two groups, one with normal, and the other with artificially-increased intracranial pressure. When suxamethonium was administered to these animals, the intracranial pressure increased in both groups, irrespective of their baseline intracranial pressure. When the same dose of suxamethonium was administered after pretreatment with thiopentone in both groups of animals, the intracranial pressure again increased from the control values.

Effect of diltiazem-induced hypotension on normal and increased intracranial pressure of cats.

The commonly used vasodilators sodium nitroprusside and nitroglycerin increase intracranial pressure (ICP) and cause tachycardia. Since diltiazem is also a vasodilator, we designed this experiment in cats to study its effect on intracranial pressure and heart rate (HR). Twelve cats were assigned to two equal groups. One group had normal ICP (N-ICP), while in the other the ICP was raised artificially (AR-ICP) by placing a balloon into the intracranial extradural space. Both groups received an infusion of diltiazem to decrease the mean blood pressure (BP), which was maintained 30 per cent below the baseline value for 15 minutes. Diltiazem caused no significant change in ICP (5.7 +/- 1 to 6.7 +/- 1.5 mmHg, p less than 0.01) in cats with N-ICP while in cats with AR-ICP, the increase from 26.9 +/- 0.5 to 34.0 +/- 1.9 mmHg was significant (p less than 0.006). HR decreased significantly during the diltiazem infusion in both groups.