Phase I study of axitinib combined with paclitaxel, docetaxel or capecitabine in patients with advanced solid tumours.

BACKGROUND: Axitinib, a potent and selective second-generation inhibitor of vascular endothelial growth factor receptors, enhanced the efficacy of chemotherapy in human xenograft tumour models. This phase I study investigated the safety, tolerability, pharmacokinetics and antitumour activity of axitinib combined with chemotherapy. METHODS: A total of 42 patients with advanced solid tumours received a continuous axitinib starting dose of 5 mg twice daily (b.i.d.) plus paclitaxel (90 mg m(-2) weekly), docetaxel (100 mg m(-2) every 3 weeks) or capecitabine (1000 or 1250 mg m(-2) b.i.d., days 1-14). RESULTS: Common treatment-related adverse events across all cohorts were nausea (45.2%), hypertension (45.2%), fatigue (42.9%), diarrhoea (38.1%), decreased appetite (33.3%) and hand-foot syndrome (31.0%). There was one complete response, nine partial responses and seven patients with stable disease. Ten patients (23.8%) remained on therapy for >8 months. Paclitaxel and capecitabine pharmacokinetics were similar in the absence or presence of axitinib, but docetaxel exposure was increased in the presence of axitinib. Axitinib pharmacokinetics were similar in the absence or presence of co-administered agents. CONCLUSIONS: Axitinib combined with paclitaxel or capecitabine was well tolerated; no additive increase in toxicities was observed. Antitumour activity was observed for each treatment regimen and across multiple tumour types.

Phase I trial of axitinib combined with platinum doublets in patients with advanced non-small cell lung cancer and other solid tumours.

BACKGROUND: This phase I dose-finding trial evaluated safety, efficacy and pharmacokinetics of axitinib, a potent and selective second-generation inhibitor of vascular endothelial growth factor receptors, combined with platinum doublets in patients with advanced non-small cell lung cancer (NSCLC) and other solid tumours. METHODS: In all, 49 patients received axitinib 5 mg twice daily (b.i.d.) with paclitaxel/carboplatin or gemcitabine/cisplatin in 3-week cycles. Following determination of the maximum tolerated dose, a squamous cell NSCLC expansion cohort was enroled and received axitinib 5 mg b.i.d. with paclitaxel/carboplatin. RESULTS: Two patients experienced dose-limiting toxicities: febrile neutropenia (n=1) in the paclitaxel/carboplatin cohort and fatigue (n=1) in the gemcitabine/cisplatin cohort. Common nonhaematologic treatment-related adverse events were hypertension (36.7%), diarrhoea (34.7%) and fatigue (28.6%). No grade ≥3 haemoptysis occurred among 12 patients with squamous cell NSCLC. The objective response rate was 37.0% for patients receiving axitinib/paclitaxel/carboplatin (n=27) and 23.8% for patients receiving axitinib/gemcitabine/cisplatin (n=21). Pharmacokinetics of axitinib and chemotherapeutic agents were similar when administered alone or in combination. CONCLUSION: Axitinib 5 mg b.i.d. may be combined with standard paclitaxel/carboplatin or gemcitabine/cisplatin regimens without evidence of overt drug-drug interactions. Both combinations demonstrated clinical efficacy and were well tolerated.

Cell cycle dependence of chloride permeability in normal and cystic fibrosis lymphocytes.

Cystic fibrosis (CF) is a genetic disease characterized by abnormal regulation of epithelial cell chloride channels. Nonepithelial cells, including lymphocytes and fibroblasts, may exhibit a similar defect. Two independent techniques were used to assess the macroscopic chloride permeability (PCl) of freshly isolated B lymphocytes and of B and T lymphocyte cell lines. Values for PCl increased specifically during the G1 phase of the cell cycle and could be further enhanced by increasing intracellular adenosine 3',5'-monophosphate (cAMP) or calcium. In lymphocytes from CF patients, regulation of PCl during the cell cycle and by second messengers was absent. Characterization of the cell cycle-dependent expression of the chloride permeability defect in lymphocytes from CF patients increases the utility of these cells in the analysis of the functional consequences of mutations in the CF gene.

PU.1 regulates the expression of the human neutrophil elastase gene.

PU.1 is a transcription factor present in B-cells and macrophages. Here, we report our studies on the role of PU.1 in myelopoiesis using human neutrophil elastase (HNE) as a model. HNE, a component of the primary granules of mature granulocytes, is a serine protease which is transcriptionally restricted to the late promyelocytic stage of granulocytic maturation. The first 200 bp of the HNE promoter directs myeloid specific expression of a reporter gene and a 30-bp element within this region was been identified as the major determinant of myeloid specific expression [S. Srikanth, T. Rado, A 30-bp element is responsible for the myeloid specific activity of the human neutrophil elastase promoter, J. Biol. Chem. 269 (1994) 32626-32632.]. We now show that the B-cell and macrophage specific transcription factor, PU.1, binds to the PU.1 consensus site within the 30-bp element to activate transcription. Substitution mutations within this recognition sequence results in the loss of PU.1 binding and in a 90% decrease in promoter activity in myeloid cells. Cotransfection of PU.1 and a reporter gene controlled by the HNE promoter into non-myeloid HeLa cells resulted in activation of reporter gene transcription.

Expression of granulocyte colony-stimulating factor by human cell lines.

We have isolated and expressed a cDNA clone that encodes a human granulocyte colony-stimulating factor from the MIA PaCa-2 cell line. A genomic clone of this factor has been isolated from the CHU-2 cell line and is reported to encode two alternative transcripts [The EMBO J. 5,575, 1986]; one transcript predicts an amino acid sequence identical to that predicted by our MIA PaCa-2 cDNA clone; the other transcript predicts a similar protein containing a three amino acid residue insertion. To investigate which types of this colony-stimulating factor are produced by other cell lines, we used specific oligonucleotides to determine which types of transcripts were present in MIA PaCa-2, 5637, and LD-1 cells, all of which have been reported to produce a factor that can stimulate the growth of predominantly granulocyte colonies in human bone marrow cell cultures. Northern analysis with these probes revealed MIA PaCa-2-like transcripts in all of these cell lines and failed to detect transcripts that would encode the colony-stimulating factor that contained the three-amino-acid-residue insertion.

Mutation in the negative regulatory element of the erythropoietin receptor gene in a case of sporadic primary polycythemia.

A 42-year-old Caucasian male with sporadic primary polycythemia has been followed by us for 13 years. During the time of observation, his hemoglobin had been stable, and he has never had an elevated white count or platelet count or any other stigmata of polycythemia vera (PV). Both of his parents, his three children, and all siblings have been hematologically normal. The in vitro culture of erythroid progenitors revealed an absence of autonomous erythropoietin (Epo)-independent erythroid colonies but demonstrated a marked increase in the sensitivity of erythroid progenitors to Epo. We have undertaken a study designed to determine whether a mutation in the Epo receptor (Epo-R) gene could cause the polycythemia phenotype seen in either dominant or recessive primary polycythemia described by us and others, or in polycythemia vera. We have sequenced the cytoplasmic positive and negative regulatory domains of the Epo-R genomic DNA, and a transversion of C to T in nucleotide 6148 was found in one of the patient's chromosomes. This mutation is located in the negative regulatory domain and results in a change from proline to serine (P488S). We have subsequently analyzed more than 40 chromosomes from unrelated normal subjects, as well as autosomal dominant, recessive, and sporadic primary polycythemia and polycythemia vera subjects. In no instance was the same or any other mutation in the Epo-R found. To determine if this Epo-R mutation is a cause of increased sensitivity of erythroid progenitors to erythropoietin, Ba/F3 cells (interleukin-3-dependent murine lymphoid line) were transfected with normal and mutated Epo-R cDNA, rendering the transfected cells viable and able to proliferate in Epo. Transfectants with wild-type and mutant Epo-R cDNA exhibited no difference in the presence of Epo. More recently, we were able to obtain DNA from the seven family members of the propositus and found that the nonpolycythemic mother and one of the siblings have the same Epo-R mutation. We conclude that this first described mutation of Epo-R encountered in humans does not appear on its own to explain the polycythemia phenotype; however, the possibility that it may interact with some other acquired or congenital abnormality in generating the polycythemia phenotype cannot be excluded.

Production of granulocyte colony-stimulating factor by a human melanoma cell line.

We have isolated a human melanoma line (LD-1) from a patient with melanoma and unexplained leukocytosis. The LD-1 cells produced a colony-stimulating factor (CSF) which stimulated primarily granulocytic colonies in human and murine bone marrow cultures. Erythroid burst and mixed colony-stimulating activity was not detected. A single CSF species with a molecular weight of 21,000 was detected in LD-1-conditioned media by G-200 chromatography. Nude mice transplanted with LD-1 tumors developed granulocytosis and had increased blood CSF levels. Messenger RNA from LD-1 cells directed the synthesis of CSF by Xenopus oocytes. Northern blots of LD-1 RNA hybridized strongly with oligonucleotide probes based on the published sequences for human G-CSF, but not with a probe based on the human GM-CSF sequence. Northern blots hybridized with an oligonucleotide probe based on the CSF-1 sequence showed a high-molecular weight band; however, low-molecular weight CSF-1 mRNAs, which are present in the CSF-1-producing cell line MIA-PaCa-2, were not detected in the LD-1 mRNA. The CSF activity of LD-1 cells is best described as human granulocyte CSF.

Differential expression of two sodium channel subtypes in human brain.

Two partial human brain sodium channel cDNA sequences (designated HBSC I and II) have been cloned and mapped to chromosome 2q23-2q24 by chromosome microdissection-PCR (CMPCR). The distribution of HBSC I and II mRNA in human brain was studied by means of a novel approach based on the ligase detection reaction. These studies demonstrate that HBSC I and II mRNA is heterogeneously distributed in brain, and that the relative ratio of the two forms can vary as much as 7-fold between different regions.

Current approaches to the molecular and physiological basis of cystic fibrosis.

Cystic fibrosis (CF) is the most common disease caused by a single gene abnormality within the caucasian population. Its severity of expression in homozygotes varies widely, and the disease involves multiple organ systems. During the past few years, major advances in CF research have been made. These advances have occurred primarily in the fields of physiology and molecular genetics. As a result of these advances, it is now generally accepted that the basic defect in CF is the inability of an epithelial chloride channel to respond to adrenergic stimulation in affected organs. The recent major breakthrough in CF research is the localization of the CF gene and identification of the mutation responsible for the majority of cases of CF. In this article, the evidence which has led to this conclusion, as well as possible mechanisms by which a mutation in a single codon can produce the CF defects are reviewed. Finally, new approaches to the characterization of the CF gene by complementation of the defect in immortal cell lines displaying the transport phenotype associated with CF are discussed.

Bone marrow injury in lithographers exposed to glycol ethers and organic solvents used in multicolor offset and ultraviolet curing printng processes.

Prompted by referral of a printer with aplastic anemia, a study of possible marrow toxicity of workplace substances was undertaken. Dermal and respiratory exposures to dipropylene glycol monomethyl ether, ethylene glycol monoethyl ether, and a range of aliphatic, aromatic and halogenated hydrocarbons used for offset and ultraviolet cured multicolor printing were documented. Evaluation of seven co-workers revealed normal peripheral blood pictures, but bone marrow specimens demonstrated clear patterns of injury in three while the others had nonspecific signs of marrow effect. These changes could not be explained by known risk factors. The authors conclude that further evaluation of possible bone marrow toxicity resulting from exposure to glycol ethers and ultraviolet curing printing processes is warranted. More generally, we have provided data demonstrating that peripheral blood counts may be an insensitive tool for the study of hematologic toxins acting at the bone marrow level.

A 30-base pair element is responsible for the myeloid-specific activity of the human neutrophil elastase promoter.

Human neutrophil elastase (HNE), a serine protease, is expressed only in the promyelocytic stages of granulocyte maturation. We examined several regions of the promoter for transcriptional activity and report that a 30-base pair (bp) element located between -76 and -106 in the 5'-flanking region of HNE is sufficient for myeloid-specific expression of HNE. Gel shift assays using nuclear extracts from myeloid and non-myeloid cells reveal several myeloid-specific complexes binding to the 30-bp element. Examination of DNA-protein interactions shows that at least two myeloid-specific proteins of 38 and 55 kDa bind to this element. DNase I protection analysis reveals two distinct footprints between -80 to -91 and -94 to -104 within this element. Transient expression studies using deletion constructs of the HNE 5'-flanking region show that the 30-bp element is active in myeloid cells K 562 and U 937 but not in HeLa cells. Internal deletion of this element results in a 60-85% loss of promoter activity in myeloid cells. Additional functional studies also show that a 19-bp region between -112 and -131 contributes to transcriptional activity of the elastase promoter as well.

Ribosomal competence and spore germination in Fusarium solani.

Extracts prepared from macroconidia of Fusarium solani f. sp. phaseoli are capable, under defined conditions, of incorporating phenylalanine into polypeptide with exogenous polyuridylic acid as messenger. Extracts from ungerminated and germinated spores have approximately the same activity. With endogenous template, leucine incorporation occurs, but in this reaction extracts from germinated spores have about 10 times more activity than do those from ungerminated spores. It is suggested that the low rate in ungerminated spores is attributable to a relative deficiency in the number of ribosomes which are organized into polysomes.

Expression of the human neutrophil elastase gene: positive and negative transcriptional elements in the 5' flanking region.

The structure and position of cis-acting DNA sequences which regulate tissue specific expression of the human neutrophil elastase (HNE) gene have been investigated. We have identified a positive and a negative regulatory element upstream from the promoter region. The ability of these sequences to regulate transcription in myeloid and non-myeloid cells was studied by inserting varying lengths of HNE 5'-flanking sequence into a reporter plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene. CAT activity in U937 was minimal in the absence of promoter and in the presence of HNE sequence to -102 bp. Inclusion of sequence up to -153 bp resulted in a 5.6-fold increase in CAT activity that was not observed in non-myeloid transfectants. Extension of the insert to include additional HNE sequence to -196 bp resulted in a decrease in CAT activity to control levels.

Phage type of tubercle bacilli isolated from patients with two or more sites of organ involvement.

To evaluate the possibility of separate pulmonary infections in human beings by different strains of Mycobacterium tuberculosis, a search for multiple phage types within a single host was under-taken. Culture isolates from 2 or more distinct anatomic sites of infection in the same patient were obtained from 87 persons. In 3 subjects, 2 distinct phage types were found. The possible explanations for 2 types in the same patient and the epidemiologic implications are discussed.

Isolation of lactoferrin cDNA from a human myeloid library and expression of mRNA during normal and leukemic myelopoiesis.

Lactoferrin is a major constituent of polymorphonuclear leukocyte granules and is present in mature neutrophils but not in blasts or promyelocytes. We have isolated a cDNA probe for lactoferrin and used it to study the synthesis of lactoferrin mRNA by normal and leukemic granulocyte precursors. The probe pHL-41 has been subcloned in phage m13 and characterized by restriction endonuclease analysis and nucleic acid sequencing. pHL-41 contains approximately 40% of the coding sequence of the lactoferrin gene. The 3' untranslated region includes a stop codon and a possible polyadenylation signal. There is a greater than 98% agreement between the cDNA-deduced amino acid sequence and that determined by analysis of the protein. Myeloid cells from normal bone marrow and circulating leukocytes from patients with chronic granulocytic leukemia contain lactoferrin mRNA transcripts that are indistinguishable in size and relative quantity. The human promyelocytic leukemia cell line HL-60 contains no lactoferrin mRNA. Induction of monocytic or granulocytic differentiation fails to induce the synthesis of detectable lactoferrin message. Similarly, studies with the human myeloblastic leukemia cell line PLB-985 reveal the inability of these cells to produce lactoferrin mRNA even under conditions that bring about morphologically demonstrable granulocytic differentiation. These data suggest that granulocytic differentiation in the leukemic cell lines is incomplete or defective. The presence of lactoferrin may play a role in the orderly expression of the genetic program leading to the development of the normal mature granulocyte.

Evidence for host-dependent modification and restriction of bacteriophage DNA in Mycobacterium tuberculosis.

Wild isolates of Mycobacterium tuberculosis may be divided into the three internationally recognized phage types on the basis of susceptibility to mycobacteriophages DS6A, BK1 and D34. Strains of type A are lysed at high efficiency by DS6A only; type B is lysed by BK1 grown on Mycobacterium smegmatis ATCC607 and DS6A, while type C is lysed additionally by D34 grown on atypical Mycobacterium F130. Propagation of D34 on a C-strain (D34-C) or BK1 on a B-strain (BK1-B) has no effect on viral host-range. D34-C has an efficiency of plating (e.o.p.) of 10(-5) on type B strains and 10(-7) on A strains. BK1-B plates on A strains at an e.o.p. of 10(-5). BK1 recovered from and repropagated on an A strain (BK1-A) has an e.o.p. of 1-0 on strains of all classes. D34-B has an e.o.p. of 1-0 on strains of type B and C, while D34-A plates with high efficiency on types B and C and displayed an e.o.p. of 10(-4) on type A. Repropagation of these viruses on the M. tuberculosis strains originally lysed by them results in the restoration of their previous host range. Variations in plating efficiency cannot be explained by differences in viral absorption alone. These findings suggest that the three phage types of human tubercle bacilli are related by a hierarchical pattern of DNA restriction and modification in which the C pattern is included in the B, and both patterns are included in A-modified DNA. Viruses such as DS6A which are equally virulent for strains of all classes are not susceptible to host dependent restriction.

Regulation of human bone marrow lactoferrin and myeloperoxidase gene expression by tumor necrosis factor-alpha.

Lactoferrin (LF) and myeloperoxidase (MPO) are glycoproteins synthesized in early myeloid cells (promyelocytes, myelocytes) and stored in granules of polymorphonuclear neutrophilic granulocytes. Both proteins are involved in the host inflammatory response, and LF has been found to have myelosuppressive activity in vivo and in vitro. Little is known, however, about the regulation of their production. We investigated the stability of LF and MPO mRNA and the effects of purified recombinant human TNF-alpha on LF and MPO levels in normal human bone marrow. Low density human bone marrow cells were cultured in the presence or absence of actinomycin D (10 micrograms/ml) or TNF-alpha (200 U/ml). LF and MPO RNA levels were analyzed by Northern blots using, respectively, a 650-bp insert from the plasmid pHL41, and a 2.3-kb insert from the plasmid pMPO2 as probes. It was found that: 1) LF mRNA is a fairly stable molecule, with a half-life of between 8 and 9 h, whereas MPO is less stable, with a half-life of between 4 and 5 h; 2) TNF-alpha decreases both LF and MPO mRNA levels, an effect seen by 24 h with MPO mRNA and 48 h with LF mRNA; 3) nuclear run-on assays revealed that TNF decreases transcription of the LF gene by 70% and the MPO gene by 50%; and 4) the suppressive effect of TNF-alpha on LF and MPO mRNA levels is not due to cell killing or selective differentiation and is reversible.

Direct amplification of a single dissected chromosomal segment by polymerase chain reaction: a human brain sodium channel gene is on chromosome 2q22-q23.

We have devised a general strategy for gene mapping based upon the direct amplification of a target sequence within a single microdissected Giemsa-banded chromosomal segment using the polymerase chain reaction. The usefulness of this approach was demonstrated by mapping a cloned human brain sodium channel (alpha subunit) gene sequence to chromosome 2q22-q23. When DNA from single, dissected chromosome segments 2q21-qter and 2q24-pter were used as templates, a sodium channel-specific 172-base-pair polymerase chain reaction product was obtained. This product was not synthesized when segments 2q21-pter and 2q24-qter were used. Chromosome microdissection-polymerase chain reaction is not only a simple, fast, and accurate method for gene mapping but also may offer significant advantages for other applications, such as cancer cytogenetics and linkage analysis.

Expression of cloned human lactoferrin in baby-hamster kidney cells.

Human lactoferrin was expressed from a cloned cDNA introduced into mammalian cells in tissue culture. Total RNA was extracted from human bone marrow, and lactoferrin cDNA was synthesized by primer-specific polymerase chain reaction after oligo(dT)-primed first-strand synthesis. The cDNA was sequenced to confirm its identity with previously published human lactoferrin sequences and cloned into the eukaryotic expression vector pNUT. Recombinant vector DNA containing the human lactoferrin sequence was introduced into baby-hamster kidney (BHK) cells in culture, and stable transfectants were produced by dominant marker selection. Human lactoferrin was expressed from the metallothionein promoter of pNUT by Zn2+ induction. The protein was secreted into the tissue-culture medium and was subsequently purified to homogeneity in a single step. Initial characterization suggests that the protein expressed by BHK cells is identical with native human lactoferrin.