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Background: 4-[2-(2-Amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-l-glutamic acid (AG2034), is a classical antifolate shown to be an excellent inhibitor of glycinamide ribonucleotide formyltransferase (GARFT), ultimately inhibiting de novo purine synthesis. We examined some metabolic effects of this drug in prostate cancer cells, LNCaP, versus non-tumorigenic prostatic epithelial cells, RWPE-1., Methods and Results: Cells were cultured in medium containing 10 nM 5-methyl-tetrahydrofolate supplemented with/without 1.7 microM hypoxanthine/1.5 microM thymidine. Cytotoxicity of AG2034 was determined by clonogenic assays. Total ATP was quantified by reverse-phase HPLC and [(14)C]-glycine incorporation and [(3)H]-hypoxanthine conversion into ATP by liquid scintillation counting. Protein expression levels were determined by Western blotting, cell cycle analysis by propidium iodide staining and cell-senescence by beta-galactosidase staining. AG2034 inhibited LNCaP cell proliferation causing death in the absence of hypoxanthine and cytostasis in its presence. However, RWPE-1 cells were resistant to AG2034 when hypoxanthine was present. AG2034 elevates AMP/ATP ratios but is unable to activate AMPK in RWPE-1 when hypoxanthine is present. Drug exposure increased expression levels of p53, p21, p27, and p16 in both cell lines and increased senescence-associated-beta-gal staining in LNCaP with/without hypoxanthine, but primarily in its absence in RWPE-1., Conclusions: LNCaP cells primarily depend upon de novo while RWPE-1 cells largely favor salvage synthesis for maintenance of their ATP pools. With AG2034 treatment, ATP synthesis via hypoxanthine salvage is insufficient to support growth of LNCaP but enough to restore ATP levels and support RWPE-1 growth. The anti-proliferative effect of AG2034 involves increasing phosphorylation of AMPK. These results indicate that AG2034 activates p53 and AMPK mediating the induction of signaling pathways leading to senescence.

Purpose: 4-[2-(2-Amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4] thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L: -glutamic acid (AG2034), is a classical antifolate, an analog of folic acid that has been shown to be an excellent inhibitor of glycinamide ribonucleotide formyltransferase (GARFT), ultimately inhibiting the de novo synthesis of purines. We examined the effect of this drug on cell proliferation, steady-state ATP levels, de novo and hypoxanthine salvage ATP synthesis, and on the phosphorylation of AMP kinase, in two different androgen independent prostate cancer cell lines, DU145 and PC-3., Methods: Cells were maintained in culture medium containing 10 nM 5-methyl tetrahydrofolate supplemented with or without 1.7 microM hypoxanthine and 1.5 microM thymidine. Cytotoxicity of AG2034 was determined by clonogenic assays. AG2034-induced inhibition of cell proliferation was determined by electronic counting of cells over varying periods of time. Total cellular AMP and ATP pre- and post-drug treatment was quantified by reverse-phase HPLC. [(14)C]-Glycine incorporation and [(3)H]-hypoxanthine conversion into ATP were determined by liquid scintillation counting of HPLC isolated ATP fractions. The phosphorylation of AMP kinase (AMPK) was detected by western blotting., Results: In the absence of 1.7 muM hypoxanthine, AG2034 was cytotoxic to both DU145 and PC-3 cells. In its presence, the cells remained cytostatic for 14 days after which time DU145 but not PC-3 re-initiated growth that was maintained for 35 days even though steady-state levels of ATP in both cell lines remained depleted and [(14)C]-glycine incorporation into ATP was inhibited by >95%. Salvage purine synthesis as measured by incorporation of [(3)H]-hypoxanthine into ATP was maintained in both cell lines albeit to different levels. When AG2034 was added to the culture medium in the presence or absence of 1.7 microM hypoxanthine, cellular ATP levels were reduced by 80% within 24 h in both the cell lines. In the absence of hypoxanthine, the AMP/ATP ratio in PC-3 cells increased by 38% and was accompanied by a modest increase in the level of phosphorylated AMPK; no increase was observed in the presence of hypoxanthine where the AMP/ATP ratio increased by approximately 10%. Under these same culture conditions, the AMP/ATP ratio in DU145 cells in the absence of hypoxanthine increased by 60% and was accompanied by a large increase in phosphorylated AMPK. In the presence of hypoxanthine however, even though the AMP/ATP ratio increased 2.5-fold, phosphorylated AMPK levels did not increase., Conclusions: The cytostatic versus the cytotoxic effect of AG2034 on PC-3 and DU145 cells is mediated by the presence or absence, respectively, of physiological levels of hypoxanthine (1.7 muM) in the media. The ability of DU145 as opposed to PC-3 cells to proliferate in the presence of AG2034 is independent of the intracellular concentration of ATP. Activation of the AMPK signaling pathway in drug-treated PC-3 and DU145 cells is cell line dependent and independent of the AMP/ATP ratio.

DC-3F/FA3 cells (FA3) were derived from antifolate-sensitive CHL cells by selection for growth in folate-free media containing 15 pM [6S]-5CHOFH4. These cells undergo a 30-fold decrease in intracellular folates, overexpress folate receptor alpha (FR alpha) and metallothionein II, and display increased sensitivity to the dihydrofolate reductase (DHFR) targeted anti-folates methotrexate (MTX) and trimetrexate (TMTX), which can be attributed primarily to the folate pool status. Upon folate repletion by growth in 15 nM [6S]-5CHOFH4, they display a 5- and 10-fold increase in resistance to both drugs, respectively, even though folate pools are restored by only 43%. Enforced overexpression of FR alpha in transfectants cultured in nanomolar folate did not confer resistance to MTX but did support a modest 2-fold increase in resistance to TMTX. Enforced overexpression of MTII had a similar effect, but when both were overexpressed together no increase in resistance beyond that conferred by each one separately was noted, suggesting that both confer resistance to TMTX through a common downstream mechanism. Analysis of three independent low folate selected clones, FA3, FA7, and FA14, showed that each had a 5- to 6-fold increase in DHFR activity accompanied by a similar increase in DHFR protein level. However, no differences were detected in the DHFR gene copy number or in the steady-state amount of DHFR mRNA, suggesting that a posttranscriptional mechanism was responsible for the increase in DHFR expression.

Purpose: Several studies have identified amino acid residues located on the hydrophobic side of the helix that forms transmembrane domain 6 (TM6) of the ABC transporter P-glycoprotein (Pgp) as being important for function. The purpose of this study was to determine if alterations to residues on the hydrophilic side could also affect function and to determine the extent to which altering the hydrophobic nature of residues on the hydrophobic side would impair the protein., Methods: A full-length cDNA encoding wild-type Pgp1 from CHL cells was used as a template for site-directed mutagenesis. Eight different mutations, three on the hydrophilic side and five on the hydrophobic side, were prepared and transfected into drug-sensitive host cells. Wild-type transfectants served as controls. Drug resistance levels, RD50 values for cyclosporin A (CsA) and verapamil, iodoarylazidoprazosin (IAAP) photolabeling and verapamil-stimulated ATPase activity were evaluated., Results: Substitution of any one of three amino acid residues on the hydrophilic side of TM6 disrupted function and led to alterations in drug resistance, CsA sensitivity, IAAP photoaffinity labeling, and in one case verapamil-stimulated ATPase activity. Replacement of a hydrophobic residue on the hydrophobic face of the helix with increasingly hydrophilic side-chains led to functional changes, the extent of which did not correlate with the degree of side-chain hydrophilicity. Finally, while the placement of a proline residue along either face of the helix had varying effects on function, in all cases its presence interfered with verapamil-stimulated ATPase activity., Conclusions: Taken together these results indicate that both faces of TM6 mediate Pgp1 function and that the expected conformational changes resulting from proline substitutions at a variety of locations within the helix can alter the protein's enzymatic activity.

The multidrug resistant cell line DC-3F/ADII was obtained by stepwise selection for growth in actinomycin D (ActD). Compared with parental cells, it displays high resistance to ActD and vincristine and low resistance to colchicine and daunorubicin. These cells overexpress a form of P-glycoprotein (Pgp1) containing a double mutation, I837L and N839I, in transmembrane domain (TM) 9; when transfected into DC-3F, this mutation confers the DC-3F/ADII phenotype. We have shown previously that another cell line, DC-3F/ADX, also displays this phenotype and overexpresses a mutant form of Pgp1 containing a double mutation in TM6 (G338A, A339P). Hence, mutations in TM9 and TM6 are independently capable of conferring the same cross-resistance phenotype. The TM6 mutations inhibit the ability of cyclosporin A to reverse cross-resistance and to block labeling of the protein by [125I]iodoarylazidoprazosin (IAAP), whereas the TM9 mutations do not show similar effects. A chimeric protein containing both pairs of mutations confers twice the level of resistance to ActD than expected from the sum of the individual mutations, but it cannot be labeled to detectable levels with [125I]IAAP. Thus, TM9 represents a novel site that cooperates with TM6 to mediate drug resistance and [125I]IAAP labeling.

DC-3F/FA3 cells (FA3) were obtained by selection of Chinese hamster lung fibroblasts for growth in folic acid free media, supplemented with 15 pM [6S]-5-formyltetrahydrofolic acid. These cells, as a result of low level gene amplification and RNA stabilization, were found to overexpress folate receptor alpha (FR alpha) mRNA by more than five hundred fold. The expression level of the receptor, a 43 kDa GPI-linked plasma membrane glycoprotein, was found to be inversely related to changes in media folate concentrations while its steady state mRNA level remained unaffected. In low folate, the rate of receptor synthesis was found to increase by more than three fold, while its half-life stabilized as compared to that observed in high folate media. Although DC-3F cells were found to contain low amounts of FR alpha mRNA, receptor expression was undetectable, and changing media folate concentrations had no effect on the expression of either. Hence, while selection for growth in low folate leads to stable overexpression of FR alpha mRNA, receptor expression is regulated at the level of protein synthesis by a mechanism sensitive to media folate levels., (Copyright 2001 Wiley-Liss, Inc.)

The impact of basal (non-induced) expression levels of metallothionein I and II on the growth of mouse embryo fibroblasts in standard DMEM/F-12 containing 8.8 microm folic acid, and in DMEM/F12 without hypoxanthine, thymidine or folic acid, containing 15 nm or 15 pm[6S]-folinic acid, was assessed by comparing wild-type MT (+/+) and homozygous null MT (-/-) cell lines. No difference in growth rate was observed between the two in DMEM/F12, although MT (-/-) cells displayed a 6-fold decrease in p27(Kip1), a two fold increase in p53 and a slight increase in p21(Waf1). After 6 days in culture, the growth rate for MT (-/-) cells in 15 nm or 15 pm[6S]-folinic acid was half that of MT (+/+). After an additional 6 days in 15 n m folate, both MT (+/+) and (-/-) cells maintained their respective growth rates, while those in 15 pm had ceased to grow. During the initial 6 days in 15 nm folate, neither cell population displayed an increase in apoptosis or a change in cell cycle distribution, even though MT (-/-) cells sustained an additional 4-fold increase in p21(Waf1)and a 6-fold decrease in cyclin E expression. At day 12, however, the MT (-/-) population, but not MT (+/+), underwent a 7-fold increase in apoptosis coupled with a 3 fold increase in S phase cells. Hence, the basal level of MT I and II constitutively expressed in MT (+/+) cells enhances growth in 15nM [6S]-folinic acid by preventing S phase arrest and apoptosis., (Copyright 2001 Academic Press.)

DC-3F/FA3 (FA3) cells, selected for growth in folic acid-free medium containing dialyzed serum and 15 pM [6S]-folinic acid, and parental DC-3F cells were compared by mRNA differential display to identify genetic changes occurring during selection. One of the genes found to be overexpressed in FA3 cells was metallothionein II (MT-II). Northern blots using a full-length hamster MT-II cDNA probe that recognizes both MT-I and MT-II RNA showed that the steady-state level of MT mRNA was elevated at least 10-fold in FA3 cells and in two other selected clones, FA7 and FA14, as well. Southern blot analysis of HindIII-digested genomic DNA indicated that amplification of neither the MT-I nor MT-II gene had occurred, and measurements of MT mRNA decay rates in the presence of actinomycin D suggested that no changes in its half-life had taken place. Hence, overexpression was due to an increase in transcription from the normal gene complement. In FA3 cells, the MT mRNA expression level was found to be directly sensitive and inversely proportional to media folate concentrations, whereas in DC-3F cells it was not, suggesting that MT gene expression is differentially regulated in these two cell lines. Overexpression of MT-II in transfected DC-3F cells was unable to support growth in 15 pM folinic acid. However, when plated in 15 nM folinic acid, a growth rate similar to FA3 cells was observed, whereas sham-transfected controls and double transfectants expressing antisense MT-II RNA and control levels of MT-II protein ceased to grow. Hence, overexpression of MT-II provides a growth advantage in low folate.

Purpose: P-glycoprotein (P-gp), a product of the Chinese hamster pgpl gene, confers multidrug resistance to mammalian cells in which it is overexpressed either by transfection or as a result of drug selection. It is encoded by a 4.3-kb mRNA and in its unglycosylated form has a predicted molecular weight of approximately 141 kDa. When a cDNA containing this sequence is transfected into drug-sensitive Chinese hamster lung cells and is expressed under the control of the beta-actin promoter, both the full-length 4.3-kb mRNA and a 2.3-kb transcript are produced. The latter results from a splicing event that utilizes near consensus 5' and 3' splicing signals resident in the full-length mRNA, and it has also been found to be present in cell lines that express the native gene. Therefore, it is a splicing product of pgpl per se. This report is concerned with the biological relevance of this transcript., Methods: In vitro transcription and translation experiments were used to show that the putative open reading frame of the 2.3-kb transcript encodes a novel 57-kDa protein (p57pgp1) that contains transmembrane domains 9-12 and the C-terminal ATP binding fold of P-gp. To elucidate the function of p57pgp1, expression vectors containing cDNAs representing (1) the 2.3-kb transcript, (2) the full-length 4.3-kb mRNA, and (3) a splice-disabled 4.3-kb transcript in which production of the 2.3-kb transcript is eliminated by an in-frame mutation at the 3' splice site, were constructed and transfected into DC-3F cells. Additional expression vectors in which p57pgp1 represented the N-terminus of a green fluorescent protein fusion construct were also prepared and used for transient expression studies., Results: Overexpression of the 2.3-kb transcript alone did not confer multidrug resistance. Transfectants in which both the 4.3-kb transcript and the 2.3-kb transcript were present, compared with transfectants in which no 2.3-kb transcript was expressed, but in which the level of expression of the 4.3-kb mRNA alone was the same, showed little change in cross-resistance pattern. However, the overall level of resistance in the latter cells was increased by approximately twofold. Hence the presence of the 2.3-kb transcript was associated with a decrease in drug resistance. In vitro transcription and translation experiments and transient expression studies indicate that p57pgp1 can be expressed both in vitro and in vivo., Conclusion: These results demonstrate that a splicing variant of pgp1 contains an open reading frame capable of translation in vitro and in vivo and suggest that alterations in splicing may contribute both directly and indirectly to the overall mechanism of pgp1-mediated multidrug resistance in CHL cells.

The expression of a P-glycoprotein (Pgp1) cDNA encoding two amino acid substitutions in the sixth transmembrane domain of the protein (G338A339 to A338P339) confers a unique cross-resistance profile that displays preferential resistance to actinomycin D and diminished resistance to colchicine and daunorubicin. We report here that this multidrug-resistant phenotype is also insensitive to reversal by cyclosporin A (CsA) but not verapamil (VRP). However, the ability of VRP to increase the accumulation of [3H]vincristine is poor in both wild-type and mutant transfectants. In contrast, the accumulation of [3H]vincristine in wild-type versus mutant transfectants in the presence of CsA is dramatically increased. It is the substitution of the alanine residue at position 339 with proline that is primarily responsible for the lowered sensitivity to CsA and for the altered drug accumulation levels. Both substitutions are required to confer the unique cross-resistance profile of the double mutant, although each independently confers a specific profile of its own. These results indicate that alterations in Pgp1 structure can differentially affect the activity of CsA and VRP to mediate drug accumulation in multidrug-resistant cells and support the conclusion that the sixth transmembrane domain of the Pgp1 transporter plays important roles, in both the specificity of drug efflux and the sensitivity of the transporter to reversal agents.

Three differently sized mRNAs are expressed from each of two DHFR (encoding dihydrofolate reductase) alleles present in the Chinese hamster lung (CHL) cell line, DC-3F. The relative abundancy of the transcripts produced from each allele differs dramatically as a result of differential utilization of the multiple poly(A) sites present in the DHFR DHFR gene and a genetic polymorphism located within the third poly(A) signal of one allele. We sought to determine whether such differences in polyadenylation affect the steady-state levels of DHFR and mRNAs expressed from either allele and, in a more general sense, to ask whether differences in 3' end RNA processing in a gene containing multiple poly(A) sites affects the final level of gene expression. An SV40 promoter-based transient expression system producing chimeric cat::DHFR transcripts was developed to regenerate the in vivo mRNA polyadenylation patterns associated with each of the two DHFR alleles. The results demonstrate that the total amount of polyadenylated RNA expressed from each of these constructs in vitro is the same regardless of the differential utilization of the poly(A) signals that occurs between them. Moreover, measurement of the individual turnover rates of the DHFR mRNAs expressed in vivo from each allele, as determined by pulse-chase labeling and actinomycin D inhibition studies, revealed no significant allele-specific differences in transcript half-lives. Finally, measuring the steady-state levels of DHFR poly(A)+ mRNA in parental DC-3F cells demonstrated that both alleles are expressed to the same extent during normal growth. Thus, even though dramatic allele-specific differences in 3' end processing of DHFR transcripts occur in vivo, such differences do not appear to influence the steady-state levels of DHFR gene expression.

Two polymorphic dihydrofolate reductase (DHFR) alleles, termed 20 K and 21 K, exist in Chinese hamster lung cells. Three major transcripts of different lengths are transcribed from each allele, and the expression of these transcripts differs dramatically between the alleles as a result of differential utilization of three poly(A) sites. Transcripts from the 20 K allele are preferentially polyadenylated at the first poly(A) site, while those from the 21 K allele are preferentially polyadenylated at the third site. In this study, transient expression experiments were used to demonstrate that a 2.1 kb genomic fragment containing the three DHFR poly(A) sites is sufficient to reproduce the allele-specific polyadenylation pattern on transiently expressed CAT-DHFR transcripts in COS cells. Site-directed mutagenesis allowed identification of the sequence elements which are responsible for this allele-specific polyadenylation. These studies indicate that a single-base change in the third poly(A) signal sequence, which alters the consensus AAUAAA signal in the 21 K allele to a weak AAUAAU signal in the 20 K allele, is primarily responsible for the dramatic difference in polyadenylation between the two alleles. Thus, as a result of this single-base change in the third poly(A) signal sequence, utilization of the first poly(A) site, located 1.2 kb upstream, changes dramatically.

Two mammalian expression plasmids, each carrying a cDNA encoding a different allele of dihydrofolate reductase (DHFR) present in the Chinese hamster lung cell line DC-3F, were constructed. These simian virus 40 promoter-enhancer-based plasmids, designated pSVA75 and pSVMQ19, are identical except for a G-->A transition at nucleotide 286 of the DHFR coding sequence. Due to this change, the enzyme expressed by pSVA75 contains Asp95, while the enzyme expressed by pSVMQ19 has Asn95 [Melera et al., J. Biol. Chem. (1988) 1978-1990]. Both forms of the enzyme are catalytically equivalent and are produced to similar levels in DC-3F cells [Yu and Melera, Cancer Res. (1993) 6031-6035; H.Y., A.H. and P.W.M., in preparation]. Clonal cell lines expressing one or the other DHFR allele were obtained via transfection of DHFR- Chinese hamster ovary cells, and 74 clones of each type isolated. These were pooled and divided into 40 aliquots, each of which was then subjected to selection by growth in sequentially increasing concentrations of methotrexate (MTX). Analysis of the resulting drug-resistant populations revealed that cells producing Asp95 DHFR dominated with an overall frequency of 3:1, and therefore, under these growth conditions, display a selective advantage over those producing Asn95 DHFR. These data extend previous observations showing that independent selections of the heterozygous parent cell line DC-3F in MTX result in threefold more MTX-resistant lines overexpressing the Asp95-encoding DHFR allele than the Asn95-encoding DHFR allele.(ABSTRACT TRUNCATED AT 250 WORDS)

Resistance to the intracellular Ca2+ pump inhibitor thapsigargin (TG) is associated with overexpression of both Ca2+ transport ATPase and the multidrug resistance (mdr) transporter P-glycoprotein (pgp). This is supported by increased resistance to TG following transfection of a functional pgp1 cDNA, and reversal of TG resistance with known inhibitors of pgp function. However, pgp is unlikely to represent the only mechanism of resistance to TG. Cell lines selected for high levels of resistance to TG (250-fold) show only a 3.7-fold increase in pgp expression and a 2-fold increase in cross-resistance to other drugs of the mdr class. Overexpression of endogenous Ca2+ transport ATPase may represent a second mechanism of resistance to TG. Increased Ca2+ ATPase expression (3-fold) is seen in cells made resistant to TG, and TG resistance increases with the transfection of a specific Ca2+ ATPase cDNA into DC-3F cells. If these transfectants are then made resistant to TG, both the endogenous Ca2+ ATPase and the exogenously transfected Ca2+ ATPase become overexpressed. These studies suggest that while TG may be a substrate for pgp, acquired resistance to TG can involve alterations in both pgp and Ca2+ ATPase expression. Additional, as yet unidentified, mechanisms of resistance may be involved in resistance to TG.

Mammalian cells displaying the multidrug resistance (mdr) phenotype are refractory to the toxic effects of a group of unrelated natural product drugs, many of which are used for cancer chemotherapy. The pattern of cross-resistance can be extremely variable among independently selected cell lines, even though such cells are often exposed to only a single drug. The overexpression of P-glycoprotein (pgp), a 150-180-kDa drug efflux pump, has been shown to confer mdr to otherwise drug-sensitive cells; however, the variable nature of cross-resistance indicates that normal pgps alone are unlikely to account for all of the observed cross-resistance phenotypes. In this report, we examined possible factors contributing to cross-resistance diversity in mammalian cells. We show that drug-resistant Chinese hamster lung cells selected during relatively short periods of drug exposure in vitro (less than 6-8 weeks) routinely overexpressed endogenous pgps and predominantly showed a cross-resistance pattern that was similar to that conferred by the introduction and overexpression of the hamster wild-type pgp1 cDNA alone. Longer drug exposure periods at higher drug concentrations, however, led to the selection of cell lines with altered cross-resistance properties. Like the short term clones, these cell lines all overexpressed endogenous pgp. In one case, the altered phenotype was shown to be caused by the acquisition of point mutations within codons 338 and 339 of the pgp1 gene, leading to two adjacent amino acid substitutions within the encoded pgp. Although the basis for the remaining altered phenotypes remains unknown, these results indicate that additional genetic alterations beyond those responsible for the initial acquisition of mdr emerge in the face of increased selective pressure, thus further modifying or complementing the cross-resistance phenotype initially conferred by wild-type pgp.

Hamster cells grown in culture may, like human and mouse cells, develop multidrug resistance (MDR) when exposed to certain cytotoxic chemotherapeutic agents. Several phenotypic features that are characteristic of MDR have been described; these include (1) resistance to many structurally and functionally unrelated drugs that have different cellular targets and modes of action; (2) reversal of MDR by certain agents, including verapamil and cyclosporin A; and (3) reduced intracellular drug accumulation relative to that of drug-sensitive cells. In this report we show that the introduction and overexpression of the hamster pgp1 cDNA confers to otherwise drug-sensitive cells an MDR phenotype with these features. Moreover, pgp1 transfectants showed varying degrees of resistance to anthracycline analogues, indicating that structural analogues of commonly used anticancer agents are capable of circumventing drug resistance conferred by pgp.

The Chinese hamster lung cell line DC-3F contains two polymorphic dihydrofolate reductase (DHFR) alleles that are defined by an Asp-Asn amino acid sequence difference at position 95 in protein. Previously, we reported that the antifolate-resistant subline DC-3F/A3 overexpressed a Leu22-->Phe mutant of the Asp95 (21k) allele and that this was the basis of its resistance to methotrexate (MTX) and methasquin [P. W. Melera, J. P. Davide, C. A. Hession, and K. W. Scotto, Mol. Cell. Biol., 4: 38-48, 1984]. We now show that another independently selected antifolate-resistant subline of DC-3F, DC-3F8/A55, in addition to being severely compromised in its ability to accumulate MTX, overexpresses a Leu22-->Phe mutant form of the Asn95 (20k) allele. Characterization of purified DHFR from these cells showed that the enzyme displayed a 6-fold higher Kd for MTX (3.92 +/- 0.17 pM) than the wild type (0.58 +/- 0.10 pM), thus explaining its lowered sensitivity to drug. Unexpectedly, however, this value was 4-fold lower than that displayed by the DC-3F/A3 enzyme even though both contain the same (Leu22-->Phe) mutation and differ only at position 95. Indeed, we have also shown that the 21k and 20k wild type enzymes, both containing Leu at position 22, in fact differ by 3-fold (1.58 +/- 0.08 and 0.58 +/- 0.10 pM, respectively) in their Kd's for MTX. This demonstrates that the amino acid at position 95 has an effect on the ability of DHFR to bind MTX. On the other hand, these allelic variants are indistinguishable from each other in their catalytic properties and in their respective Kd's for dihydrofolate. Taken together, these characteristics are consistent with the observation that it is the wild type 21k allele which is preferentially overexpressed at a frequency of 3:1 in MTX-resistant Chinese hamster lung sublines derived by long-term selection in MTX. The results of these studies are novel in that they establish a role for allelic variation in the DHFR gene as a contributor to antifolate resistance in mammalian cells. Moreover, they implicate amino acid position 95 in the maintenance of the structure of the MTX binding pocket.

The Chinese hamster cell line, DC-3F, is heterozygous at the DHFR locus, and each allele can be distinguished on the basis of a unique DNA restriction pattern, protein isoelectric profile and in the abundancy of the DHFR mRNAs it expresses. Although each allele produces four transcripts, 1000, 1650 and 2150 nucleotides [corrected] in length, the relative distribution of these RNAs differs for each; the 2150 nt mRNA represents the major (60%) species generated from one allele, while the 1000 nt mRNA is the major species generated from the other. The allele that predominantly expresses the 2150 nt transcript is preferentially overexpressed when DC-3F cells are subjected to selection in methotrexate. We have analyzed the 3' ends of both DHFR alleles and have found that the three major mRNAs arise by readthrough of multiple polyadenylation signals. A four base deletion in one allele changes the consensus polyadenylation signal AAUAAA to AAUAAU, resulting in the utilization of a cryptic polyadenylation signal lying 21 bp upstream. Surprisingly, this mutation in the third polyadenylation signal appears to affect not only the utilization of this signal, but also the efficiency with which the first signal, located 1171 bp upstream from the third site, is utilized.

We have developed a modified RNase protection assay in which the antisense RNA probe is prepared from a PCR-amplified DNA template rather than from a linearized plasmid DNA template. In this assay, an RNA polymerase promoter sequence is attached to the 5' end of the antisense PCR primer. Using this modified antisense primer in conjunction with the paired sense primer, PCR amplification generates a linear DNA template that includes an RNA polymerase promoter sequence. Transcription in vitro initiated by the incorporated promoter in the presence of RNA polymerase and ribonucleotide triphosphates produces a radiolabeled run-off antisense RNA transcript, which can then be used as probe for RNase protection analysis. Probes generated by this method obviate the need to subclone DNA sequences into transcription vectors for synthesis of antisense transcripts. Due to the simplicity of its design and the lack of need for subcloning, this strategy offers greater flexibility than conventional methods for the production of single-stranded RNA probes, and thus facilitates the implementation of the ribonuclease protection assay.

Stable expression of a full-length cDNA encoding chicken fast muscle Ca2+ transport ATPase was obtained in a Chinese hamster lung cell line (DC-3F), using a dual-promoter expression vector (pH beta FCaA3) in which the ATPase was cloned downstream of a human beta-actin gene promoter, and a mutant dihydrofolate reductase cDNA (A3/DHFR) was cloned downstream of an SV40 promoter-enhancer. Owing to its essentially normal catalytic activity and modest (20-fold) resistance to the antifolate methotrexate (MTX), the A3/DHFR mutant enzyme served as an efficient dominant selection marker in transfected cell populations challenged with MTX and, within a broad range of drug concentrations, allowed subsequent amplification and overexpression of vector sequences. In stable transfectants, the expressed ATPase was targeted to intracellular membranes, and the microsomal fractions from those cells exhibited high rates of Ca2+ transport. In comparative experiments using transient expression in COS1 cells, the level of ATPase per transfected cell was greater, but less than 5% of the transfected population exhibited ATPase expression. Furthermore, as opposed to the stable lines, the transiently expressing cells could not be propagated. Overall, the yield of ATPase was 12-16 and 4-6 micrograms per milligram of microsomal protein in the stable and the transient expression systems, respectively. The advantages of the stably transfected cell lines therefore lie in the homogeneity of ATPase expression and its distribution in cells and microsomes, in the large yield of microsomes obtained by continuous cell propagation, and in the reproducible functional characteristics of the microsomes. Moreover, the microsomes derived from stably transfected cell lines provide a convenient system for studies of Ca2+ transport and ATPase partial reaction, eliminating the need to conduct repetitive transient transfections to obtain sufficient amounts of enzyme for functional studies.

Eukaryotic cells can display resistance to a wide range of natural-product chemotheraputic agents by the expression of P-glycoprotein (pgp), a putative plasma membrane transporter that is thought to mediate the efflux of these agents from cells. We have identified, in cells selected for multidrug resistance with actinomycin D, a mutant form of pgp that contains two amino acid substitutions within the putative sixth transmembrane domain. In transfection experiments, this altered pgp confers a cross-resistance phenotype that is altered significantly from that conferred by the normal protein, displaying maximal resistance to actinomycin D. These results strongly implicate the sixth transmembrane domain in the mechanism of pgp drug recognition and efflux. Moreover, they indicate a close functional homology between pgp and the cystic fibrosis transmembrane regulator in which the sixth transmembrane domain has also been shown to influence substrate specificity.

Simian virus 40 promoter-enhancer-based mammalian expression plasmids using dihydrofolate reductase (DHFR)-encoding cDNA sequences originally isolated from two methotrexate (MTX)-resistant, DHFR-overproducing Chinese hamster lung cell lines were constructed. One, designated pSVA75, contains a DHFR cDNA that encodes leucine (Leu22) and corresponds to the wild type (wt), MTX-sensitive form of the enzyme [Melera et al., J. Biol. Chem. 263 (1988) 1978-1990]. The other plasmid, pSVA3, contains a cDNA that encodes a novel mutant form of the enzyme in which Leu22 has been changed to Phe [Melera et al., Mol. Cell Biol. 4 (1984) 38-48]. The resulting DHFR displays a 20-fold-enhanced resistance to inhibition by MTX, but maintains the catalytic activity of the wt enzyme [Albrecht et al., Cancer Res. 32 (1972) 1539-1546]. Transfection of DHFR- Chinese hamster ovary cells with either plasmid demonstrated that both were able to reconstitute the DHFR+ phenotype with equal efficiency (i.e., greater than 2.5 x 10(-3), indicating that both the wt and mutant enzymes were catalytically active in transfected cells. In addition, the mutant form of the enzyme was found to act as a dominant selectable marker when transfected into diploid DHFR+ cells, and to allow selection of resistant clones at low MTX concentrations (125 nM MTX) with a frequency of greater than 8 x 10(-4). Moreover, transfected clones were found to amplify their exogenous DHFR sequences to reasonably high levels (42-fold) at relatively low (888 nM) MTX concentrations, suggesting that substantial amplification of DHFR DNA and cotransfected sequences as well, can be achieved with this vector.

The expression of resistance by tumor cells to anticancer drugs remains a primary cause of patient failure to chemotherapeutic intervention in cancer. Although studied for many years, both the acquisition and maintenance of drug resistance continue to provide active areas for research and the application of modern molecular and cell biological techniques to those questions has begun to generate a wealth of new information. This article attempts to summarize and contrast some of the more recent observations concerning acquired and intrinsic resistance to the classical antifolate methotrexate, and suggests that the mechanisms of intrinsic resistance, and to a lesser extent acquired resistance as well, may be more broad based than previously thought and that the expression of intrinsic resistance may be strongly influenced by physiological and genetic variation.

In an effort to better understand the preferential resistance to actinomycin D displayed by the multidrug-resistant Chinese hamster lung cell line DC-3F/ADX, we have cloned from those cells a number of cDNAs representing p-glycoprotein gene transcripts. Of the 12 clones isolated, all represent pgp1 transcripts and one, pADX165, contains a 4304-base pair insert with an open reading frame encoding a 1276-amino acid protein that is the homolog of the mouse mdr3/mdr1a gene product. A domain by domain comparison of this protein with p-glycoproteins capable of supporting multidrug resistance, i.e. human mdr1, mouse mdr1/mdr1b, and mouse mdr3/mdr1a, shows that, in addition to the ATP binding sites, the second, fourth, and eleventh transmembrane domains and the four small intracellular loops, IC-1, IC-2, IC-4, and IC-5, are highly conserved and are therefore likely to be important for the maintenance of p-glycoprotein function. Of the remaining 11 cDNA clones, 9 were found to be truncated versions of pADX165. Two others, however, pADX185 and pADX124, contained internal deletions resulting in open reading frames capable of encoding lnovel forms of p-glycoprotein. S1 nuclease and RNase protection analysis demonstrated that these cDNAs represent transcripts present in a number of different multidrug-resistant Chinese hamster lung cell lines. Hence, both are considered to be splicing variants of the hamster pgp1 gene primary transcript.

In multidrug resistance, which is observed clinically and in tissue culture, cells that are challenged with certain cytotoxic drugs develop resistance not only to the selective agent but also to other, seemingly unrelated, agents. The multidrug-resistant phenotype is associated with DNA sequence amplification and with the overproduction of a number of cytosolic and membrane glycoproteins. The differential amplification and altered expression of at least two related genes, termed multidrug-resistant associated genes has been shown in multidrug-resistant Chinese hamster cells. In multidrug-resistant mouse and human cells, genes homologous to those in Chinese hamster cells are also amplified. The level of expression of these genes varied and did not correlate with their copy number. Furthermore, in Chinese hamster cells, the development of resistance to a single drug and multidrug resistance were closely related, but uncoupled, events. The overexpression of the multidrug-resistant genes was better correlated with the degree of resistance to the selective agent than it was with the extent of multidrug resistance.

The parasitic protozoan Leishmania mexicana amazonensis has two developmental stages: a motile flagellated promastigote stage and a sessile intracellular amastigote stage. In our previous work, cells of the promastigote stage were found to synthesize more tubulin protein than those of the amastigote stage. Here, tubulin mRNAs in these leishmanias were analyzed. Based on dot blot hybridization between total leishmanial RNA and tubulin-specific cDNA probes derived from chicken brain, amastigotes and promastigotes were found to have approximately equal amounts of alpha- and beta-tubulin mRNAs. RNA blotting of leishmanial RNA, using chicken tubulin cDNA probes, showed that amastigotes and promastigotes both gave a single mRNA species of 2100 nucleotides for alpha-tubulin in roughly similar quantities. However, such analysis for beta-tubulin revealed mainly a single mRNA species of 3600 nucleotides for amastigotes and three species of 2800, 3600, and 4400 nucleotides for promastigotes, the smallest mRNA being the most predominant. Thus, regulation of gene expression appears to be different only for beta-tubulin between the two developmental stages of this protozoan.

We have analyzed a 3.8 kb Eco RI fragment of genomic DNA obtained from the amplified N-myc gene of human neuroblastoma cell line BE(2)-C. This fragment contains an exon with an open reading frame encoding approximately 170 amino acids of the carboxy-terminal end of the putative N-myc protein. Comparison of the inferred amino acid sequence of this peptide with that of the 3' domain of the human c-myc protein shows that locally conserved but dispersed regions of homology exist throughout the lengths of these peptides, while hydropathy plots indicate that the physical properties implied by their primary sequences are strikingly similar. Based upon these and other considerations, it is suggested that the 3' domains of c-myc and N-myc may potentially share related functions.

Purification of a cDNA probe specific for dihydrofolate reductase mRNA has allowed the relative quantification of dihydrofolate reductase gene numbers and mRNA levels between a line of Chinese hamster lung fibroblasts sensitive to the antifolate drugs methotrexate and methasquin and sublines which by virtue of their overproduction of the target enzyme dihydrofolate reductase are cross-resistant to these site-specific inhibitors. Previous results have shown that resistant sublines could overproduce one of two molecular weight forms of dihydrofolate reductase, one parental-like (Mr = 21,000) and one nonparental-like (Mr = 20,000), each encoded by a different mRNA present in elevated amounts in resistant cells (Melera, P. W., Wolgemuth, D., Biedler, J. L., and Hession, C. (1980). J. Biol. Chem. 255, 319--322). The cDNA-RNA and cDNA-DNA hybridization experiments reported here confirm the elevated levels of dihydrofolate reductase-specific mRNA in resistant cells and show that the overproduction of either molecular weight form of dihydrofolate reductase is accompanied by amplification in dihydrofolate reductase gene number.

Using a differential extraction procedure which had previously been shown to yield one nucleic acid fraction enriched in cytoplasmic RNA and another enriched in nuclear RNA, we have been able to isolate two polyadenylated RNA populations from microplasmodia of Physarum polycephalum. The poly(A)-containing RNA from the cytoplasmic-enriched fraction accounts for approximately 1.2% of the cytoplasmic nucleic acid, has a number-average nucleotide size of 1339+/- 39 nucleotides, and has been shown, in a protein-synthesizing system in vitro, to be capable of directing the synthesis of peptides which have also been shown to be synthesized in vivo by microplasmodia. The poly(A)-containing RNA from the nuclear-enriched fraction has a number-average nucleotide size of 1533 +/- 104 nucleotides and represents a mixture of cytoplasmic and nuclear adenylated RNA molecules. Based upon these observations, we have identified the polyadenylated RNA isolated from the fraction enriched in cytoplasmic nuclei acid as Physarum poly(A)-containing messenger RNA.

ds cDNA from antifolate-resistant Chinese hamster lung fibroblast subline DC-3F/MQ19 was ligated to Eco RI and Sal I oligonucleotide linkers and cloned into Eco RI and Sal I digested pBR322. Transformed colonies containing dihydrofolate reductase (DHFR)-specific recombinant plasmid were identified by Grunstein Hogness assay using a Chinese hamster DHFR-specific cDNA probe. A recombinant plasmid, pDHFR6, containing a 650 bp HFR insert was isolated and analyzed. This plasmid was used as a molecular probe in a Northern blot analysis of both cytoplasmic and polysomal DHFR, poly A+ mRNAs of the DC-3F/MQ19 subline, which over-produces a 20,000d DHFR 150-fold, and DC-3F/A3 subline, which over-produces a 21,000d DHFR 170-fold. This analysis revealed the presence of three DHFR mRNA species of 1350, 2200, and 3300 nucleotides in both independently-derived cell lines. The relative abundance of each species however varied strikingly between the two cell lines.

Six independently derived multidrug-resistant Chinese hamster cell lines selected with vincristine, daunorubicin, actinomycin D, or colchicine were probed by in situ hybridization techniques with the cloned cDNA, p5L-18, to chromosomally localize known or presumed amplified P-glycoprotein genes. One or two clusters of amplified genes were demonstrable in all of the highly resistant sublines and were localized to homogeneously staining regions and/or abnormally banding regions, gene amplification-associated cytogenetic abnormalities, on eight different chromosomes. Analysis of trypsin-Giemsa banded karyotypes revealed additional, multiple chromosomal rearrangements that were apparently nonspecific. Mapping studies localized the native P-glycoprotein gene(s) to the region q23 to 31 (most probably band 26) on the long arm of chromosome 1 of normal Chinese hamster bone marrow fibroblasts and normal chromosome 1 homologues in resistant cells. Southern blot analysis of restriction endonuclease fragments indicated the amplification of one or both of (at least) two wild-type nonallelic genes in four of the lines and the presence in one line (DC-3F/DMM XX) of a unique 5.0-kilobase BamHI fragment resulting from a recombinational event during amplification. Comparison with the cytogenetic data indicated no correlation between restriction patterns generated by EcoRI, HindIII, PstI, or BamHI and chromosomal location of amplified genes. However, the only sublines in which the homogeneously staining region or abnormally banding region is positioned at 1q26 (at or near the site of the native gene) exhibit either alterations in gene structure (DC-3F/DM XX) or in regulation of gene expression (DC-3F/AD X), suggesting a process more complex than simply amplification of the gene in loco.

Nucleotide sequence analysis of a cDNA clone shown to direct the synthesis in Escherichia coli of a pI 6.5 form of dihydrofolate reductase (DHFR) with an apparent molecular weight of 21,000 has clarified the allelic nature of the DHFR genes present in the Chinese hamster lung cell line DC-3F. By comparison with other cDNAs encoding different forms of DHFR produced by these cells or by antifolate-resistant sublines derived from them (Melera, P.W., Davide, J.P., Hession, C.A., and Scotto, K.W (1984) Mol. Cell. Biol. 4, 38-48) and with the use of transcription vectors to generate homogeneous populations of specific DHFR mRNAs for subsequent translation in vitro, we demonstrate that, with respect to the proteins they encode, these alleles differ only at amino acid position 95; a conversion of Asp----Asn at this position is solely responsible for the electrophoretic mobility and pI differences between the Mr 21,000 pI 6.5 and Mr 20,000 pI 6.7 forms of the enzyme. We also show that the conversion of Leu to Phe at position 22 of the Mr 21,000 pI 6.5 enzyme results in a mutant form whose catalytic activity is equal to or greater than normal, but whose IC50 for methotrexate is 85 microM. Additionally, the in vitro translation experiments show that the minor pI forms of DHFR known to exist in Chinese hamster lung cells are generated by a translational or post-translational modification step. Preliminary evidence suggests that this modification may result from an acetylation of the N terminus of the protein.

DNA purified from a Chinese hamster cell line of lung fibroblast origin (DC83F) was analyzed by density gradient centrifugation and by gel electrophoresis after restriction endonuclease digestion in order to fractionate discrete repetitive fractions within the total DNA. No obvious satellite DNAs were resolved using the CsCl or Ag-Cs2SO4 density gradient conditions described herein. However, analysis of the digestion products of a battery of restriction endonucleases indicated that three of these enzymes, EcoR1, HaeIII, and XhoI, yielded discrete fragments which could be visualized with EtBr staining or identified by scintillation counting of [3H]DNA. DNAs from several highly (greater than or equal to hundredfold increased resistance) antifolate-resistant sublines of DC-3F, characterized by a large homogeneously staining region (HSR) in the chromosome complement, were examined with both techniques and compared to the parental, antifolate-sensitive cell line DNA. The density gradient profiles and electrophoretic patterns of restriction endonuclease digests were identical among all the cell lines examined and were indistinguishable from those of the parental DC-3F DNA.

Southern blot analysis of a number of EcoRI-digested human neuroblastoma DNAs has revealed the presence of a family of discrete restriction fragments, the majority of which are amplified in most, but not all, of the neuroblastoma cell lines tested. None of these sequences is abundantly present in DNA from other human tumors of different tissue origins, including several either known or presumed to contain amplified DNA. Hence, these sequences appear to be specifically amplified by neuroblastoma cells. Hybridization with metaphase chromosomes in situ has localized these sequences to either the homogeneously staining regions or double-minute chromosomes of different neuroblastoma cell lines, indicating that these chromosomal structures, although present in cell lines established from different patients, share many sequences and may have a common, but as yet unknown, function.

Plasma membranes were prepared from the human lymphocyte cell line WIL23A by hypotonic swelling, Dounce homogenization, differential and equilibrium centrifugation. The resulting vesiculated membrane fragments were found to have densities of 1.10 and 1.17 g/ml, and were defined by lactoperoxidase mediated whole cell iodination, L-[3H] fucose incorporation, 5'-nucleotidase activity (EC 3.1.3.5) and electron micrographic visualization. Recovery of plasma membrane from whole cell homogenates was estimated to be approximately 30-35% as judged by the recovery of 125I-labeled cell surface protein. When plasma membranes were prepared from cells which had been incubated for 18 h in the presence of 0.5 muCi/ml [3H] thymidine such that greater than 10(9) acid insoluble counts could be demonstrated in the whole cell homogenates, no [3H] thymidine label and presumably, therefore, no DNA, could be shown to be coincident with either the 1.10 or 1.17 density. Similar experiments with [3H] uridine suggested that 90% of the plasma membranes did not contain RNA, while 10% remained questionable.

An MCF-7 human breast cancer cell line was selected which was 200-fold more resistant to Adriamycin than the wild type cell line. This Adriamycin-resistant (AdrR) cell line exhibited a multidrug-resistant phenotype and was cross-resistant to a wide range of antineoplastic agents including Vinca alkaloids, anthracyclines, and epipodophyllotoxins. Cytogenetic analysis of the AdrR cell line showed the presence of homogeneously staining regions on several chromosomes which were not present in the parental cell line. Using the technique of in-gel renaturation, DNA sequences which were amplified 50- to 100-fold in the AdrR cell line and which covered a total of over 140 kilobases were isolated. In addition, AdrR cells were found to contain amplified and overexpressed sequences which were homologous to hamster P-glycoprotein gene sequences. A hamster cDNA P-glycoprotein gene probe was used to screen a lambda gt10 cDNA library made from human AdrR cell line mRNA and human cDNA sequences homologous to the P-glycoprotein gene were isolated. Hybridization studies with the cloned human cDNA (pADR1) showed that the AdrR MCF-7 cell line contained a 60-fold amplification of this DNA sequence and that polyadenylated mRNA from the AdrR cell line contained a 4.8-kilobase transcript which was overexpressed 45-fold. There was a direct correlation between DNA and RNA copy number of this sequence and level of resistance among several MCF-7 Adriamycin-resistant cell lines. In situ hybridization studies demonstrated that the human P-glycoprotein gene sequence was found on chromosome 7q21.1 in normal human lymphocytes and that amplified DNA sequences isolated from the AdrR MCF-7 cells by the in-gel hybridization technique were linked to the human P-glycoprotein sequences in the homogeneously staining regions in the AdrR cells.

Isoacceptors of Physarum polycephalum Ala-, Arg-, Glu-, Gln-, Gly-, Ile-, Leu-, Lys-, Ser-, Thr-, and Val-tRNAs were resolved by reverse-phase chromatography and isolated, and their codon recognition properties were determined in a ribosomal binding assay. Codon assignments were made to most isoacceptors, and they are summarized along with those determined in other studies from Escherichia coli, yeasts, wheat germ, hymenoptera, Xenopus, and mammals. The patterns of codon recognition by isoacceptors from P. polycephalum are more similar to those of animals than to those of plants or lower fungi.

We carried out cytogenetic studies of four Chinese hamster, mouse, and human cell lines selected for high levels of resistance (500- to 4,000-fold) to vincristine (VCR) by a multistep selection procedure. All cells examined contained gene amplification-associated metaphase chromosome abnormalities, either homogeneously staining regions (HSRs), abnormally banding regions (ABRs), or double-minute chromosomes (DMs); control actinomycin D- and daunorubicin-resistant hamster lines did not exhibit this type of chromosomal abnormality. VCR-resistant Chinese hamster sublines exhibited both increased synthesis of the protein V19 (Mr 19,000; pl = 5.7) and increased concentrations of V19 polysomal mRNA. When VCR-resistant cells were grown in drug-free medium, level of resistance, synthesis of V19, and amount of V19 mRNA declined in parallel with mean length of the HSR or mean number of DMs per cell. Cross-resistance studies indicate that VCR-resistant cells have increased resistance both to antimitotic agents and to a wide variety of agents unrelated to VCR in chemical structure and/or mechanism of action. Our studies of tubulin synthesis in Chinese hamster cells indicate no overproduction of tubulin or presence of a mutant tubulin species. Comparison with antifolate-resistant Chinese hamster cells known to contain amplified dihydrofolate reductase genes localized to HSRs or ABRs strongly suggests that the HSRs, ABRs, or DMs of the Vinca alkaloid-resistant sublines likewise represent cytological manifestations of specifically amplified genes, possibly encoding V19, involved in development of resistance to VCR.

Purification of the dihydrofolate reductases overproduced by 16 independently-derived antifolate-resistant sublines of Chinese hamster lung cells and their drug-sensitive parental cell line, DC-3F, has confirmed our original observation that two molecular weight classes of this enzyme, viz. Mr = 21,000 can be overproduced by drug-resistant cells (Melera, P. W., Wolgemuth, D., Biedler, J. L., and Hession, C. (1980) J. Biol. Chem. 255, 319-322). Of the 16 drug-resistant sublines analyzed, 12 overproduce the 21,000-dalton enzyme, while 4 overproduce the 20,000-dalton enzyme. Both molecular weight classes are found in the drug-sensitive parental line. Two-dimensional polyacrylamide gel electrophoresis of the two molecular weight classes of enzyme has shown that each is composed of two isoelectric forms, and that the relative distributions of the isoelectric forms within a molecular weight class are maintained during overproduction. In vitro translation experiments have demonstrated that the synthesis of these isoelectric forms and their relative distributions are mediated by mRNA. Finally, evidence is presented for the existence of two sets of three dihydrofolate reductase mRNAs in overproducing sublines. Both sets, within a subline, encode the same molecular weight class of dihydrofolate reductase, while each set directs the synthesis of only one of the isoelectric forms within that class. Although the genetic basis for these observations is not yet understood, it is clear that the production of dihydrofolate reductase by Chinese hamster lung cells is complex and probably involves the expression of multiple dihydrofolate reductase genes and/or alleles.

Three independently-derived, antifolate-resistant Chinese hamster lung cell lines that exhibit low level increases in dihydrofolate reductase (DHFR) activity, i.e., three- to fivefold vs. controls, have been compared with drug-sensitive cells to determine relative DHFR gene content. With a solution hybridization technique that makes use of genomic DNA and a cloned double-stranded Chinese hamster DHFR cDNA probe, it has been found that the enzyme activity increases are associated with an approximately proportionate amplification of DHFR genes. Trypsin-Giemsa staining techniques and hybridizations in situ further show that the amplified DHFR genes are located within abnormally banding regions along chromosome 2q and also suggest that, in each subline, only one chromosome 2 homolog is initially involved in the amplification process.

Nucleotide sequence analysis of two cDNA clones, one shown to direct the synthesis in Escherichia coli of the pI 6.7 form of the 20,000-molecular-weight class of Chinese hamster lung cell dihydrofolate reductase, and the other shown to direct the synthesis of the pI 6.5 form of the 21,000-molecular-weight class of the enzyme, has revealed the following: (i) the differences in physical and enzymatic properties displayed by these two proteins are due to two variations in their respective amino acid sequences with the conversion of Leu to Phe at position 22 probably responsible for the differential sensitivity of these two enzymes to methotrexate and methasquin; (ii) the multiple mRNAs responsible for the synthesis of each of these proteins differ in size due, at least in part, to a length heterogeneity at their 3' ends; (iii) these two proteins are encoded by different genes; and (iv) the sequence AAATATA appears to be a major polyadenylation signal in one Chinese hamster lung cell dihydrofolate reductase gene and a minor signal in another.

We have begun a series of studies designed to characterize gene expression during differentiation in the slime mold Physarum polycephalum. This work concerns the starvation phase of the sporulation sequence and describes some of the quantitative changes which occur in plasmodial constituents during the 3-day starvation period and also describes alterations in the transfer ribonucleic acid (tRNA) population. The results show that whereas the plasmodial tRNA content decreased by 75% during starvation, concurrent de novo synthesis of tRNA also occurred, and they also show that overall amino acid acceptor activity of the starvation-phase tRNA population did not differ significantly from that found in the growth phase. Of the 19 starvation-phase tRNA families assayed, however, 6 were found to have consistently lower acceptor activities than did their growth-phase counterparts. Reverse-phase (RPC-5) chromatographic analysis of five of those families failed to reveal any major differences between growth- and starvation-phase isoacceptors. The data suggest that the depletion and resynthesis of tRNA during the starvation phase results in a quantitative alteration in the composition of the tRNA population and that the alteration is tRNA family and not tRNA isoacceptor specific.

The native Chinese hamster DHFR gene was localized to 2pter----q14 using a combination of somatic cell hybrid technology and molecular techniques. In addition, PGD, ENO1, and DTS, previously mapped to Chinese hamster chromosome 2, were localized to 2q14----qter. Localization of PGM1 to this region was confirmed. The relationship of the site of the native DHFR gene to the location of the homogeneously staining region in methotrexate-resistant cells is discussed.

Screening of a partial cDNA library prepared from the human neuroblastoma cell line BE(2)-C with genomic DNA probes containing sequences representative of the amplified domain of that cell line allowed us to identify cloned transcripts from an active gene within the domain. The gene BE(2)-C-59 is amplified ca. 150-fold and encodes a 3.0- and a 1.5-kilobase RNA transcript, both of which are overproduced in BE(2)-C cells. A survey of a large variety of human tumor cell types indicated that this gene is amplified to varying degrees in all neuroblastoma cell lines and a retinoblastoma cell line that exhibit obvious cytological manifestations of DNA sequence amplification, i.e., homogeneously staining regions and double-minute chromosomes. The BE(2)-C-59 gene is not amplified, however, in other nonrelated tumor types, even those containing amplified DNA. Although the functional significance of this specific gene amplification in neuroblastoma cells remains unknown, an indication that it may relate to the malignant phenotype of these cells follows from the remainder of our data which show that the amplified BE(2)-C-59 gene shares partial homology with both the second and third exons, but not the first exon, of the human c-myc oncogene.

A comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins synthesized by antifolate-sensitive (DC-3F) and -resistant (DC-3F/A3 and DC-3F/MQ19) Chinese hamster lung fibroblast cells has shown that an apparent molecular weight difference exists between the dihydrofolate reductases overproduced by the resistant sublines. Translation in a rabbit reticulocyte lysate system of polysomal and cytoplasmic mRNAs from the three sublines has shown that the overproduction of dihydrofolate reductase by resistant cells is accompanied by increased levels of dihydrofolate reductase-specific mRNA and that the different molecular weight forms of dihydrofolate reductase are encoded by their respective mRNAs.