Modulation of drug resistance via ubiquitin carboxy-terminal hydrolase

The expression of ubiquitin carboxy-terminal hydrolase is aberrent in cells that are resistant to treatment with chemical agents. Accordingly, the invention features methods for diagnosing and treating drug resistant cells (e.g., tumor cells) by examining and modulating the expression or activity of UCH.

BACKGROUND OF THE INVENTION 
The field of the invention is modulation of drug resistance. 
The concentrations of proteins in biological cells are regulated by elegant 
biochemical mechanisms. By way of these mechanisms, cells eliminate 
damaged proteins and can, by altering the concentrations of biologically 
active proteins, such as enzymes, alter cellular processes that are 
important for the overall well being of the organism. 
In eukaryotic cells, proteins can be selectively degraded via the ubiquitin 
pathway. Ubiquitin is a highly conserved protein that is covalently 
ligated to proteins in a process referred to as ubiquitination. Proteins 
that have been ubiquitinated are committed to degradation by a 26S 
protease complex. The ubiquitin pathway is thought to play an important 
role in regulating cellular processes by regulating protein levels. 
Numerous review articles have described various aspects of the ubiquitin 
pathway. For example, the molecular genetics of the ubiquitin system have 
been reviewed by Finley et al. (Ann. Rev. Cell Biol. 7:25-69, 1991) and 
Jentsch et al. (Biochim. Biophys. Acta 1089:127-139, 1991); the 
involvement of the system in pathological states has been reviewed by 
Mayer et al. (Biochim. Biophys. Acta 1089:141-157, 1991); and the 
biochemistry and enzymology of various stages of the ubiquitin pathway 
have been reviewed by Hershko and Ciechanover (Ann. Rev. Biochem. 
61:761-807, 1992). Interest in the ubiquitin pathway is due in part to the 
wide variety of physiological processes that are affected by 
ubiquitination of proteins. These processes include the heat shock 
response, DNA repair, cell cycle progression, the modification of histones 
and of receptors, and the possible pathogenesis of selected 
neurodegenerative diseases. 
Only the protein conjugated to ubiquitin is degraded via the proteasome; 
ubiquitin itself is recycled by ubiquitin carboxy-terminal hydrolases, 
which cleave the bond between ubiquitin and the protein targeted for 
degradation. These enzymes constitute a family of thiol proteases, and 
homologues have been found in, for example, yeast (Miller et al., 
BioTechnology 7:698-704, 1989; Tobias and Varshavsky, J. Biol. Chem. 
266:12021-12028, 1991; Baker et al., J. Biol. Chem. 267:23364-23375, 
1992), bovine (Papa and Hochstrasser, Nature 366:313-319, 1993), avian 
(Woo et al., J. Biol. Chem. 270:18766-18773, 1995), Drosophila (Zhang et 
al., Dev. Biol. 17:214, 1993) and human (Wilkinson et al., Science 
246:670, 1989) cells. 
SUMMARY OF THE INVENTION 
As described herein for the first time, the expression of ubiquitin 
carboxy-terminal hydrolase (UCH; sometimes abbreviated UCTH) is aberrent 
in cells that are resistant to treatment with chemical agents, for 
example, pharmaceutical agents. Accordingly, the invention features 
methods for diagnosing and treating drug resistant cells (e.g., tumor 
cells) by examining and modulating the expression or activity of UCH. The 
cells can be resistant to one or more pharmaceutical agents, and so may, 
as appropriate, be referred to as drug resistant or multidrug resistant 
cells. 
The present invention provides a method of assessing expression, especially 
aberrant expression, of a cellular UCH gene, which may indicate the 
presence, persistence, or reappearance of tumor cells in an individual's 
tissue. UCH gene expression is assessed by obtaining a sample of cellular 
tissue from a mammal (e.g., a human), preferably from a site in the body 
suspected of containing malignant tissue, and obtaining RNA from the 
tissue. The RNA is combined with a UCH oligonucleotide under standard 
conditions for hybridization, and the resulting mixture is assayed for the 
presence of hybrids consisting of the UCH oligonucleotide and a cellular 
UCH gene transcript. As further described below, the oligonucleotide can 
be detectably labeled or otherwise modified. For example, the 
oligonucleotide can have a peptide-nucleic acid backbone. The presence 
and/or relative abundance of hybrids indicates aberrant expression of a 
cellular UCH gene, and correlates with the occurrence of transformed cells 
in situ, especially transformed cells having a drug-resistant phenotype. 
Information obtained by practicing the diagnostic methods described herein 
will be useful in determining the prognosis for patients with malignancies 
(tumors) that are characterized by expression of UCH and thus by a 
drug-resistance phenotype. The information will also assist the clinician 
in designing chemotherapeutic or other treatment regimes to eradicate such 
malignancies from the body. The methods described herein can be practiced 
with a sample of any body tissue type, such as that from the mammary, 
respiratory, urogenital, endocrine, or immune systems. The present methods 
are particularly useful in assessing biopsy tissue obtained from the 
breast, respiratory system (e.g., by bronchoalveolar lavage), ovary, 
uterus, cervix, prostate, testes, pancreas, spleen, bone marrow, or 
lymphatic system. 
In addition, the invention features therapeutic compositions, such as those 
containing a compound that modulates expression of UCH protein, and 
therapeutic methods in which these compounds are administered to a 
patient. Accordingly, the invention provides means for mitigating 
(detectably decreasing or otherwise affecting) aberrant expression of a 
UCH gene or protein, and for attenuating an undesirable drug-resistance 
phenotype, particularly a phenotype contributed by UCH. More particularly, 
the invention features methods for mitigating aberrant expression of a UCH 
gene, and/or aberrant activation or alteration of a UCH polypeptide. One 
embodiment involves the administration of a pharmaceutical composition 
containing an antisense UCH oligonucleotide to a mammal suffering from the 
effects of altered expression and/or function of UCH. Another embodiment 
involves the administration of an antibody or fusion polypeptide that 
specifically binds UCH. In either embodiment, the therapeutic agent is 
administered systemically or locally under conditions sufficient to 
mitigate or attenuate the phenotype associated with aberrant expression or 
activity of UCH (including undesirably high or undesirably low levels of 
UCH expression or activity). Preferably, the therapeutic agent is 
administered under conditions sufficient to destroy cells producing 
aberrant levels or UCH or cells in which the activity of UCH is higher 
than that in a comparable cell. For example, either of the foregoing 
therapeutic agents can be administered as an adjunct to conventional 
chemotherapy. That is, either of the foregoing therapeutic agents can be 
coadministered together with one or more chemotherapeutic drugs. In this 
manner, the invention provides for destruction of drug-resistant tumor 
cells in situ. The present antisense or fusion polypeptide therapeutic 
agent can be administered prior to, concomitant with, or following 
administration of one or more chemotherapeutic drugs. In such embodiments, 
the antisense pharmaceutical composition mitigates resistance of 
UCH-expressing cells to the cytotoxic effects of the chemotherapeutic 
drug. That is, the antisense composition attenuates the phenotype 
associated with an aberrant level of UCH expression or activity, which is 
characterized by the property of drug resistance. This is accomplished by 
disrupting activation or transcription of the UCH gene, or by 
destabilizing RNA transcripts thereof. Diminished or discontinued 
expression of UCH renders cells more susceptible to the cytotoxic effects 
of a chemotherapeutic drug. Similarly, a therapeutically administered 
cytotoxic fusion polypeptide localized in the vicinity of cells aberrantly 
expressing UCH can produce cytolysis thereof. Alternatively (or in 
addition), a chemoattractant fusion polypeptide localized to 
UCH-expressing cells can stimulate their destruction by macrophages, 
killer T cells, or cytotoxic T cells. 
Another aspect of the invention features methods for identifying a 
modulator of UCH. In general, this aspect of the invention relies on the 
use of a UCH-expressing host cell. Prokaryotic or eukaryotic host cells 
can be used for purposes of identifying a UCH inhibitor. However, in 
general, eukaryotic host cells are preferred. Yeast or mammalian cells can 
be used, as desired or as dictated by specific circumstances, as can plant 
cells. Presently, mammalian host cells, particularly human cells are 
preferred. The UCH-expressing host cell is contacted with a candidate 
modulator, and after a sufficient period of time for modulatory effects 
(inhibition or stimulation) to be manifested, the cell is assayed to 
determine whether the candidate modulator indeed affects UCH. In one 
embodiment, the level of cellular UCH gene expression is assayed. A 
detectable decrease in (attenuation or abrogation) or cessation of UCH 
gene expression indicates that the candidate modulator is a UCH inhibitor. 
Another embodiment involves assay of the amount or rate of production of 
UCH polypeptide by the cell. A detectable decrease or cessation of 
immunologically recognized UCH polypeptide indicates that the candidate 
modulator is a UCH inhibitor. In another embodiment, the host cell is 
contacted with a substrate (e.g., a cytotoxin) degraded by the ubiquitin 
pathway. The candidate inhibitor is contacted with the host cell prior to, 
concomitantly with, or following exposure to the substrate. The amount of 
substrate degraded by the cell is assessed. A detectable decrease in 
degradation of the substrate indicates that the candidate is a UCH 
inhibitor. Alternatively, in specific embodiments wherein the substrate is 
cytotoxic, survival of the host cell is assessed. A detectable decrease in 
survival indicates that the candidate is an UCH inhibitor. Candidate 
substances appropriate for screening as UCH modulators in any of the 
foregoing embodiments include natural or synthetic metabolites, toxins, 
antibiotics, elements of a combinatorial chemistry, nucleotide, or peptide 
library, naturally sourced cell secretion products, cell lysates, and the 
like. 
An additional aspect of the invention features a UCH modulator identified 
by any of the above-described methods. Preferably, the modulator is a 
small molecule, for example, an element of a combinatorial chemistry 
library or a low molecular weight natural or synthetic product or 
metabolite. The modulator may be dispersed in a pharmaceutically 
acceptable vehicle to produce a drug-resistance attenuating pharmaceutical 
composition of the present invention. 
Another aspect of the invention thus features inhibitor-based methods of 
mitigating aberrant UCH expression and/or polypeptide production. The 
present method involves the step of administering a UCH inhibitor, 
optionally dispersed in a pharmaceutically acceptable vehicle to a mammal 
suffering from effects of a UCH-associated aberrancy. In a preferred 
embodiment, the invention provides a method for improving (potentiating) 
effectiveness of chemotherapy to eradicate UCH-expressing cells, for 
example, drug-resistant transformed cells, from the body of a mammal. This 
method involves the steps of administering a chemotherapeutic drug to the 
mammal, and coadministering a UCH inhibitor identified as described 
herein. Preferably, the inhibitor is provided in the form of a 
drug-resistance attenuating composition, i.e., dispersed in a 
pharmaceutically acceptable vehicle. This method is particularly preferred 
where a chemotherapy adjunct is desired to eradicate drug-resistant tumor 
cells. Advantageously, the method can be practiced where a fluid tumor 
(e.g., leukemia, lymphoma, lymphosarcoma or ascites) is present, or where 
the situs of a primary or metastatic tumor is deemed unsuitable for 
surgical intervention or, especially, where a remontant or reemergent 
tumor is observed following an initial course of chemotherapeutic 
treatment. 
The term "modulation" as used herein refers to both upregulation (i.e., 
activation or stimulation), for example by agonizing, and downregulation 
(i.e., inhibition or suppression), for example by antagonizing of a 
bioactivity (e.g., expression of a gene). 
The term "an aberrant activity" or "abnormal activity," as applied to an 
activity of a protein, refers to an activity which differs from the 
activity of the wild-type or native protein or which differs from the 
activity of the protein in a healthy subject. An activity of a protein can 
be aberrant because it is stronger than the activity of its native 
counterpart. Alternatively, an activity can be aberrant because it is 
weaker or absent relative to the activity of its native counterpart. An 
aberrant activity can also be a change in an activity. For example, an 
aberrant protein can interact with a different protein relative to its 
native counterpart. A cell can have an aberrant activity due to 
overexpression or underexpression of the gene encoding UCH. 
"Biological activity" or "bioactivity" or "activity" or "biological 
function," which are used interchangeably for the purposes herein, when 
applied to UCH, mean an effector or antigenic function that is directly or 
indirectly performed by a UCH polypeptide (whether in its native or 
denatured conformation), or by any fragment thereof. A biological activity 
is also intended to include binding to a protein, such as binding to a 
domain of UCH. UCH bioactivity can be modulated by directly affecting UCH 
protein. Alternatively, UCH bioactivity can be modulated by modulating the 
level of UCH protein, such as by modulating expression of a UCH gene. 
Antigenic functions include possession of an epitope or antigenic site 
that is capable of cross-reacting with antibodies raised against a 
naturally occurring or denatured UCH polypeptide or fragment thereof. 
The term "treating" as used herein is intended to encompass curing as well 
as ameliorating at least one symptom of a condition or disease. 
Unless otherwise defined, all technical and scientific terms used herein 
have the same meaning as commonly understood by one of ordinary skill in 
the art to which this invention belongs. Preferred materials and methods 
are described below. However, those of ordinary skill in the art will 
understand that materials and methods that are similar or equivalent to 
those described herein can be used in the practice or testing of the 
present invention. 
All publications, patent applications, patents, and other references 
mentioned herein are incorporated by reference in their entirety. Features 
and advantages of the invention will be apparent from the following 
detailed description, and from the claims.

DETAILED DESCRIPTION 
Mammalian cells having a "drug-resistant" or "multidrug-resistant" 
phenotype are characterized by the ability to export or expel or otherwise 
desa plurality of cytotoxic substances (e.g., chemotherapeutic drugs) from 
the intracellular milieu. Cells can acquire this phenotype as a result of 
selection pressure imposed by exposure to a single chemotherapeutic drug 
(the selection toxin). Alternatively, cells may exhibit the phenotype 
prior to toxin exposure, since the export of cytotoxic substances may 
involve a mechanism in common with normal export of cellular secretion 
products, metabolites, and the like. Multidrug resistance differs from 
simple acquired resistance to the selection toxin in that the cell 
acquires competence to export or otherwise dispose of additional 
cytotoxins (other chemotherapeutic drugs) to which the cell was not 
previously exposed. For example, a multidrug-resistance cell population 
was isolated by culturing the H69 cell line, derived from a human small 
cell lung carcinoma, in the presence of adriamycin as a selection toxin 
(Mirski et al., Cancer Res. 47:2594-2598, 1987). 
As shown in the example below, UCH is highly expressed in cells that are 
resistant to cytotoxic agents. A cell is said to be resistant to a 
cytotoxic agent if it is unaffected by that agent, or less affected than a 
similar cell (e.g., a transformed cell of a given phenotype may be less 
affected than a non-transformed cell of the same phenotype). It follows 
that one can predict whether a cell will be resistant to treatment with a 
chemical agent by examining the level of UCH expression or activity, and 
further, that one can modulate cellular resistance to chemical agents by 
altering the expression or activity of UCH. 
Monitoring UCH protein production or gene expression levels, or 
fluctuations therein, in one or more tumor biopsy samples will provide 
information relevant to the diagnosis, prognosis and/or staging of 
neoplastic disease in a cancer sufferer. Any suitable means for detecting 
UCH protein production or gene expression can be applied. Preferably, 
diagnosis is achieved by hybridization techniques involving the use of a 
modified UCH probe, as further described below. A particularly preferred 
technique involves the use of a peptide-nucleic acid probe as described, 
for example in Egholm et al. (Nature 365:566-568, 1993) and Perry-O'Keefe 
et al. (Proc. Natl. Acad. Sci. USA 93:14670-14675, 1996). 
A Wide Variety of Cell Types are Amenable to the Diagnostic and Therapeutic 
Methods Described Herein 
The diagnostic and therapeutic methods described below can be applied to 
any cell that expresses UCH, i.e., to any eukaryotic cell. Furthermore, 
any cell type that can be made either more or less resistant to a chemical 
agent by altering the level of UCH expression or activity can be used in 
the practice of the invention. A wide variety of cell types are 
encompassed because UCH is an essential and highly conserved enzyme that 
functions in a fundamental biochemical pathway used by all eukaryotic 
cells. 
The cell types used in the practice of the invention can be, for example, 
fungal cells (such as a yeast cell), plant cells (such as those of an 
edible crop, a fruit-bearing tree, or a decorative plant), or higher 
eukaryotic cells (such as the cells of a mammal) that exhibit an altered 
response to a fungicide, herbicide, or chemotherapeutic agent, 
respectively. 
Furthermore, mammalian cells that are amenable to the diagnostic and 
therapeutic methods described herein include a wide variety of cell types 
that proliferate uncontrollably and thereby give rise various cancers. For 
example, the diagnostic and therapeutic methods of the invention can be 
applied to cancerous cells of mesenchymal origin, such as those producing 
sarcomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, 
osteogenic sarcoma or chordosarcoma, angiosarcoma, endotheliosarcoma, 
lymphangiosarcoma, synoviosarcoma or mesotheliosarcoma); leukemias and 
lymphomas such as granulocytic leukemia, monocytic leukemia, lymphocytic 
leukemia, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, or 
Hodgkins disease; sarcomas such as leiomysarcoma or rhabdomysarcoma, 
tumors of epithelial origin such as squamous cell carcinoma, basal cell 
carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, 
adenocarcinoma, papillary carcinoma, papillary adenocarcinoma, 
cystadenocarcinoma, medullary carcinoma, undifferentiated carcinoma, 
bronchogenic carcinoma, melanoma, renal cell carcinoma, hepatoma-liver 
cell carcinoma, bile duct carcinoma, cholangiocarcinoma, papillary 
carcinoma, transitional cell carcinoma, choriocarcinoma, semonoma, or 
embryonal carcinoma; and tumors of the nervous system including glioma, 
meningoma, medulloblastoma, schwannoma or epidymoma. Additional cell types 
amenable to diagnosis and treatment according to the methods described 
herein include those giving rise to mammary carcinomas, gastrointestinal 
carcinoma, such as colonic carcinomas, bladder carcinoma, prostate 
carcinoma, and squamous cell carcinoma of the neck and head region. The 
diagnostic and therapeutic methods of the invention can be carried out 
using a mammary cell, or a cell in the respiratory, urogenital, endocrine, 
or immune system. 
Diagnostic Assays: Resistance to Chemical Agents Can be Correlated with UCH 
Expression or Activity 
Determining the most effective therapeutic regime for a particular disease 
or condition can be difficult. For example, patients who have developed a 
cancer or other disease associated with the proliferative growth of cells 
must, together with their physician, decide between treatment regimes that 
are based on chemotherapy, radiation therapy, or surgery. It would, 
therefore, be useful to determine whether the proliferating cells are 
likely to be resistant or sensitive to chemotherapeutic agents. The 
discovery that UCH is upregulated in drug resistant cells provides the 
basis for diagnostic methods that can be used to determine whether the 
cells affected in any given disease or condition are likely to be 
resistant to chemical agents. The method can be carried out, for example, 
by assessing the expression or activity of UCH in the cells in question. 
An elevated level of expression or activity (for example, in comparison 
with the level of expression or activity in a non-tumor cell) would 
indicate the presence of a disease or condition in which cells are 
resistant to chemical agents, such as chemotherapeutic agents. Conversely, 
a normal or low level of expression or activity would indicate the 
presence of a disease or condition in which cells are sensitive to 
chemical agents. 
To perform the diagnostic methods, a sample of tissue is first obtained. 
The most appropriate tissues to be sampled, when given a particular set of 
symptoms, are known to those of skill in the art of medicine, particularly 
those who routinely diagnose and treat patients with cancer. The sample 
itself can be any sample containing diseased cells, such as a blood, 
urine, serum, or plasma sample. Alternatively, the sample can be a tissue 
sample (e.g., biopsy tissue), or an effusion obtained from a joint (e.g., 
synovial fluid), from the abdominal cavity (e.g., ascites fluid), from the 
chest (e.g., pleural fluid), from the central nervous system (e.g., 
cerebral spinal fluid), or from the eye (e.g., aqueous humor). The sample 
can also consist of cultured cells that were originally obtained from a 
patient (e.g., peripheral blood mononuclear cells). In many instances, the 
sample will be obtained from a human patient, but can also be obtained 
from a plant, or from an animal that is valued for commercial worth (e.g., 
a cow, sheep, or pig), for athletic performance (e.g., dogs or horses that 
race), or for companionship (e.g., a cat, dog, or other pet). 
Those of skill in the art are readily able to determine whether cells 
within a sample contain a normal or aberrant amount of UCH, and whether 
that expression correlates with an increased resistance to the action of a 
chemical agent. This determination can be made by following the protocol 
set forth in the example below, in which RNA is harvested from cells that 
have an undesirably high level of resistance to a chemotherapeutic agent 
and is assessed by Northern blot analysis. Alternatively, the level of UCH 
gene expression can be assessed by performing a quantitative PCR-based 
assay (the experimental basis being set forth in Mullis, K. B., 1987, U.S. 
Pat. No. 4,683,202), an RNAse protection assay, or in situ hybridization. 
To obtain a probe suitable for assessing UCH mRNA expression, those of 
ordinary skill in the art can obtain the known sequence of a UCH-encoding 
nucleic acid molecule from publications (e.g., see Day et al., FEBS Lett. 
210:157-160, 1987) or by searching the GenBank database accessible through 
the Internet address: http://www.ncbi.nlm.nih.gov/). To move directly to 
the sequence of a human mRNA for UCH (formerly referred to as protein gene 
product (PGP) 9.5), those of ordinary skill in the art can search GenBank 
using the Accession Number X04741. 
If desired, the expression of UCH can also be assessed at the level of 
translation, where UCH protein, rather than the mRNA encoding it, is 
analyzed. Typically, protein expression is analyzed by Western blot 
analysis or, in situ, by immunohistochemistry. Alternatively, the activity 
of UCH can be assessed in vitro, for example by performing assays 
analogous to those described by woo et al. in assessing UCHs isolated from 
chick skeletal muscle (J. Biol. Chem. 270:18766-18733, 1995). 
The levels of UCH expression or activity in cells collected from a patient 
can be compared with cells harvested from a non-affected region of the 
patient's body, or from a healthy individual. Cells within a sample (e.g., 
malignant cells within biopsy tissue) that overexpress UCH are likely to 
be resistant to chemotherapeutic agents. In this circumstance, the level 
of UCH expression or activity can be altered, as described below, in order 
to alter the resistance of the cell to chemotherapeutic agents: lowering 
the level of UCH expression or activity should lower the resistance of the 
cell to therapeutic agents. 
Furthermore, two or more biopsy samples can be obtained from an individual 
(e.g., a cancer sufferer) and assessed at different times. For example, a 
first biopsy sample corresponds to a time of diagnosis or to a time prior 
to or concomitant with the onset of chemotherapy, and a second biopsy 
sample can correspond to a timepoint at which beneficial results of 
chemotherapy are expected to be detectable (e.g., a time sufficiently 
remote from the onset of chemotherapy for cytotoxic effects to be 
observed). One or more subsequent biopsy samples may correspond to further 
timepoints optionally correlated with fluctuations in clinical parameters 
(e.g., relapse, remission, a change in disease staging, or the like). 
Changes in UCH expression are expected to correlate with, or to predict, 
the emergence or attenuation of a drug-resistance phenotype. 
It will be appreciated that the causes of drug-resistance phenotypes vary 
with each individual cell type and may not be wholly accounted for by 
expression or overexpression of UCH. 
Decreasing the Resistance of a Cell to a Chemical Agent 
The resistance of a cell to a chemical agent can be lowered (i.e., the cell 
can be made more sensitive to the effects of a chemical agent, such as a 
pharmaceutical agent), by decreasing the expression or activity of UCH. 
Skilled artisans are readily able to determine the circumstances in which 
it would be desirable to decrease the resistance of a cell to a cytotoxic 
or otherwise deleterious chemical agent. Generally, these circumstances 
include any situation in which it is desirable to treat a cell with a 
chemical agent that is otherwise ineffective. For example, it would be 
desirable to lower the resistance of cancerous cells to chemotherapeutic 
agents in order to more effectively treat the cancer. The resistance may 
be lowered in any cell, regardless of the level of expression of UCH. 
Thus, cells that express normal levels of UCH are also candidates for 
treatment. 
Decreasing UCH Expression with Antisense Oligonucleotides 
Therapeutic methods that are based on the administration of antisense 
oligonucleotides require synthesis of oligonucleotides (either DNA or RNA) 
that are complementary to UCH mRNA molecules; the antisense 
oligonucleotides will specifically bind to the complementary UCH mRNA 
transcripts and prevent translation. 
Absolute complementarity, although preferred, is not required. A sequence 
"complementary" to a portion of an RNA, as referred to herein, means a 
sequence having sufficient complementarity to be able to hybridize with 
the RNA, forming a stable duplex; in the case of double-stranded antisense 
nucleic acid molecules, a single strand of the duplex DNA may thus be 
tested, or triplex formation may be assayed. The ability to hybridize will 
depend on both the degree of complementarity and the length of the 
antisense nucleic acid. Generally, longer antisense oligonucleotides can 
contain mismatches with their targets yet still form a stable duplex (or 
triplex, as the case may be). One of ordinary skill in the art can 
ascertain a tolerable degree of mismatch by using standard procedures to 
determine the melting point of the hybridized complex. Antisense 
oligonucleotides complementary to mRNA coding regions are less efficient 
inhibitors of translation than oligonucleotides that are complementary to 
5'- or 3'-untranslated sequence, but could be used in accordance with the 
instant invention. The antisense nucleic acids should be at least six 
nucleotides in length, and are preferably oligonucleotides ranging from 6 
to about 50 nucleotides in length. In specific aspects, the 
oligonucleotide is at least 10 nucleotides, preferably at least 17 
nucleotides, more preferably at least 25 nucleotides, and most preferably 
at least 50 nucleotides in length. 
Regardless of the choice of target sequence, it is preferred that in vitro 
studies are first performed to quantitate the ability of the antisense 
oligonucleotide to inhibit gene expression. It is preferred that these 
studies utilize controls that distinguish between antisense gene 
inhibition and nonspecific biological effects of oligonucleotides. It is 
also preferred that these studies compare levels of the target RNA or 
protein with that of an internal control RNA or protein. Additionally, it 
is envisioned that results obtained using the antisense oligonucleotide 
are compared with those obtained using a control oligonucleotide. It is 
preferred that the control oligonucleotide is of approximately the same 
length as the test oligonucleotide and that the nucleotide sequence of the 
oligonucleotide differs from the antisense sequence no more than is 
necessary to prevent specific hybridization to the target sequence. 
The oligonucleotide can be modified at the base moiety, sugar moiety, or 
phosphate backbone, for example, to improve stability of the molecule, 
hybridization, etc. The oligonucleotide may include other appended groups 
such as peptides (for example, for targeting host cell receptors in vivo), 
or agents facilitating transport across the cell membrane (see, for 
example, Letsinger et al., Proc. Natl. Acad. Sci. USA 86:6553-6556, 1989; 
Lemaitre et al., Proc. Natl. Acad. Sci. USA 84:648-652, 1987; PCT 
Publication No. WO88/09810, published Dec. 15, 1988), or the blood-brain 
barrier (see, for example, PCT Publication No. W089/10134, published Apr. 
25, 1988), hybridization-triggered cleavage agents. (see, for example, 
Krol et al., BioTechniques 6:958-976, 1988) or intercalating agents (see, 
for example, Zon, Pharm. Res. 5:539-549, 1988). To this end, the 
oligonucleotide can be conjugated to another molecule, for example, a 
peptide, hybridization triggered cross-linking agent, transport agent, 
hybridization-triggered cleavage agent, and the like. 
The antisense oligonucleotide may comprise at least one modified base 
moiety that is selected from the group including, but not limited to, 
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, 
xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 
5-carboxymethylaminomethyl-2-thiouridine, 
5-carboxymethylaminomethyluracil, dihydrouracil, 
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 
2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 
7-methylguanine, 5-methylaminomethyluracil, 
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 
5'-methoxycarboxymethyluracil, 5-methoxyuracil, 
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), 
wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, 
uracil-5-oxyacetic acid methylester, uracil-5oxyacetic acid (v), 
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 
and 2,6-diamiaminopurine. 
The antisense oligonucleotide can also comprise at least one modified sugar 
moiety selected from the group including, but not limited to, arabinose, 
2-fluoroarabinose, xylulose, and hexose. 
In yet another embodiment, the antisense oligonucleotide comprises at least 
one modified phosphate backbone selected from the group consisting of a 
phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a 
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl 
phosphotriester, and a formacetal or analog thereof. 
In yet another embodiment, the antisense oligonucleotide is an 
.alpha.-anomeric oligonucleotide. An .alpha.-anomeric oligonucleotide 
forms specific double-stranded hybrids with complementary RNA in which, 
contrary to the usual .beta.-units, the strands run parallel to each other 
(Gautier et al., Nucl. Acids Res. 15:6625-6641, 1987). The oligonucleotide 
can be a 2'-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 
15:6131-6148, 1987), or a chimeric RNA-DNA analogue (Inoue et al., FEBS 
Lett. 215:327-330, 1987). 
Oligonucleotides used in the practice of the invention can be synthesized 
by standard methods known in the art, for example, by use of an automated 
DNA synthesizer (such as are commercially available from Biosearch, 
Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides 
can be synthesized by the method of Stein et al. (Nucl. Acids Res. 
16:3209, 1988), and methylphosphonate oligonucleotides can be prepared by 
use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. 
Acad. Sci. USA 85:7448-7451, 1988). 
The antisense molecules would be delivered to cells that overexpress UCH in 
vivo. A number of methods have been developed for delivering antisense DNA 
or RNA to cells; for example, antisense molecules can be injected directly 
into the tissue site. Alternatively, modified antisense molecules have 
been designed that target the desired cells (for example, antisense 
molecules can be linked to peptides or antibodies that specifically bind 
receptors or antigens expressed on the target cell surface). These target 
specific molecules can be administered systemically. 
It can be difficult to achieve intracellular concentrations of antisense 
molecules that are sufficient to suppress translation of endogenous mRNAs. 
Therefore, a preferred approach utilizes a recombinant DNA construct in 
which the antisense oligonucleotide is placed under the control of a 
strong pol III or pol II promoter. The use of such a construct to 
transfect target cells in the patient will result in the transcription of 
sufficient amounts of single stranded RNAs that will form complementary 
base pairs with the endogenous UCH transcripts and thereby prevent 
translation of UCH mRNA. For example, a vector can be introduced in vivo 
such that it is taken up by a cell and directs the transcription of an 
antisense RNA. Such a vector can remain episomal or become chromosomally 
integrated, as long as it can be transcribed to produce the desired 
antisense RNA. 
Methods of designing antisense oligonucleotides and introducing them into 
host cells have been described in, for example, Weinberg et al. (U.S. Pat. 
4,740,463; hereby incorporated by reference). For an antisense 
oligonucleotide that has been demonstrated to specifically inhibit UCH 
expression, see Maki et al. (Differentiation 60:59-66, 1996). 
Decreasing UCH Expression with Ribozyme Molecules 
Ribozyme molecules designed to catalytically cleave UCH mRNA transcripts 
can also be used to prevent translation of UCH mRNA and expression of UCH. 
(See, e.g., PCT International Publication WO90/11364, published Oct. 4, 
1990; Sarver et al., Science 247:1222-1225, 1990). While ribozymes that 
cleave mRNA at site specific recognition sequences can be used to destroy 
UCH mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead 
ribozymes cleave mRNAs at locations dictated by flanking regions that form 
complementary base pairs with the target mRNA. The sole requirement is 
that the target mRNA have the following sequence of two bases: 5'-UG-3'. 
The construction and production of hammerhead ribozymes is well known in 
the art and is described more fully in Haseloff and Gerlach, Nature 
334:585-591, 1988. There are many potential hammerhead ribozyme cleavage 
sites within the nucleotide sequence of human UCH cDNA. Preferably, the 
ribozyme is engineered so that the cleavage recognition site is located 
near the 5' end of the UCH mRNA; i.e., to increase efficiency and minimize 
the intracellular accumulation of non-functional mRNA transcripts. 
Ribozymes useful in the present invention also include RNA 
endoribonucleases (hereinafter "Cech-type ribozymes") such as the one 
which occurs naturally in Tetrahymena Thermophila (known as the IVS, or 
L-19 IVS RNA) and which has been extensively described by Thomas Cech and 
collaborators (Zaug et al., Science 224:574-578, 1984; Zaug and Cech, 
Science 231:470-475, 1986; Zaug et al., Nature 324:429-433, 1986; 
published International patent application No. WO 88/04300 by University 
Patents Inc.; Been and Cech, Cell 47:207-216, 1986). The Cech-type 
ribozymes have an eight basepair active site that hybridizes to a target 
RNA sequence, whereafter cleavage of the target RNA takes place. 
As in the antisense approach, the ribozymes can be composed of modified 
oligonucleotides (e.g. for improved stability, targeting, etc.) and should 
be delivered to cells that express UCH in vivo, for example, cells within 
a tumor or benign growth. A preferred method of delivery involves using a 
DNA construct "encoding" the ribozyme under the control of a strong 
constitutive pol III or pol II promoter, so that transfected cells will 
produce sufficient quantities of the ribozyme to destroy endogenous UCH 
messages and inhibit translation. Because ribozymes, unlike antisense 
molecules, are catalytic, a lower intracellular concentration is required 
for efficiency. 
Decreasing UCH Expression by Homologous Recombination 
Endogenous UCH gene expression can also be reduced by inactivating or 
"knocking out" the UCH gene or its promoter using targeted homologous 
recombination (e.g., see Smithies et al., Nature 317:230-234, 1985; Thomas 
and Capecchi, Cell 51:503-512, 1987; Thompson et al., Cell 5:313-321, 
1989). For example, a mutant, non-functional UCH (or a completely 
unrelated DNA sequence) flanked by DNA homologous to the endogenous UCH 
gene (either the coding regions or regulatory regions) can be used, with 
or without a selectable marker and/or a negative selectable marker, to 
transfect cells that express UCH in vivo. Insertion of the DNA construct, 
via targeted homologous recombination, results in inactivation of the UCH 
gene. The recombinant DNA constructs can be directly administered or 
targeted to the required site in vivo using appropriate viral vectors, for 
example, herpes virus vectors for delivery to brain tissue, where 
inoperable tumors can arise. 
Alternatively, endogenous UCH gene expression can be reduced by targeting 
deoxyribonucleotide sequences complementary to the regulatory region of 
the UCH gene (i.e., the UCH promoter and/or enhancers) to form triple 
helical structures that prevent transcription of the UCH gene in target 
cells in the body. (See generally, Helene, Anticancer Drug Des. 6:569-84, 
1991; Helene et al., Ann. N.Y. Acad. Sci. 660:27-36, 1992; and Maher, 
Bioassays 14:807-15, 1992). 
Decreasing UCH Activity with Antibodies or Chemical Compounds 
The invention can also be practiced using antibodies that specifically bind 
UCH. Antibodies that specifically recognize one or more epitopes of UCH 
enzymes include, but are not limited to, polyclonal antibodies, monoclonal 
antibodies (mAbs), humanized or chimeric antibodies, single chain 
antibodies, Fab fragments, F(ab').sub.2 fragments, fragments produced by a 
Fab expression library, anti-idiotypic (anti-Id) antibodies, and 
epitope-binding fragments of any of the above. 
Antibodies can be used, for example, to detect UCH in a biological sample 
and may, therefore, be utilized as part of a diagnostic or prognostic 
technique whereby patients may be tested for abnormal amounts of UCH (as 
described above). Additionally, such antibodies can be used in conjunction 
with gene therapy techniques to, for example, evaluate cells expressing 
UCH following transfection with a UCH-encoding nucleic acid sequence. 
Preferably, the antibodies recognize epitopes of UCH that are unique, 
i.e., are not present on other proteins, even if those proteins function 
in the ubiquitin pathway or are otherwise related to UCH. 
Monoclonal antibodies, which are homogeneous populations of antibodies to a 
particular antigen, may be obtained by any technique that provides for the 
production of antibody molecules by continuous cell lines in culture. 
These include, but are not limited to, the hybridoma technique of Kohler 
and Milstein (Nature 256:495-497, 1975; and U.S. Pat. No. 4,376,110), the 
human B cell hybridoma technique (Kosbor et al., Immunology Today 4:72, 
1983; Cole et al., Proc. Natl. Acad. Sci. USA 80:2026-2030, 1983), and the 
EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies And Cancer 
Therapy," Alan R. Liss, Inc., pp. 77-96, 1985). Such antibodies may be of 
any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any 
subclass thereof. The hybridoma producing mabs useful in the practice of 
this invention can be cultivated in vitro or in vivo. Due to production of 
high titers of mabs, in vivo production is generally preferred. 
In addition, techniques developed for the production of "chimeric 
antibodies" (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855, 
1984; Neuberger et al., Nature 312:604-608, 1984; Takeda et al., Nature 
314:452-454, 1985) by splicing the genes from a mouse antibody molecule of 
appropriate antigen specificity together with genes from a human antibody 
molecule of appropriate biological activity can be used. A chimeric 
antibody is a molecule in which different portions are derived from 
different animal species, such as those having a variable region derived 
from a murine mAb and a human immunoglobulin constant region. 
Alternatively, techniques described for the production of single chain 
antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-426, 1988; 
Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; and Ward et 
al., 1989, Nature 334:544-546, 1989) can be adapted to produce single 
chain antibodies against UCH gene products. Single chain antibodies are 
formed by linking the heavy and light chain fragments of the Fv region via 
an amino acid bridge, resulting in a single chain polypeptide. 
Antibody fragments that recognize specific epitopes can be generated by 
known techniques. These fragments include but are not limited to: the 
F(ab').sub.2 fragments which can be produced by pepsin digestion of the 
antibody molecule, and the Fab fragments which can be generated by 
reducing the disulfide bridges of the F(ab').sub.2 fragments. 
Alternatively, Fab expression libraries may be constructed (Huse et al., 
Science 246:1275-1281, 1989) to allow rapid and easy identification of 
monoclonal Fab fragments with the desired specificity. These antibodies 
can, in turn, be utilized to generate anti-idiotype antibodies that 
"mimic" UCH, using techniques well known to those skilled in the art. 
(See, for example, Greenspan and Bona, FASEB J. 7:437-444, 1993; and 
Nissinoff, J. Immunol. 147:2429-2438, 1991). 
Antibodies can be humanized by methods known in the art. For example, 
monoclonal antibodies with a desired binding specificity can be 
commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, 
Calif.). Fully human antibodies, such as those expressed in transgenic 
animals are also features of the invention (Green et al., Nature Genetics 
7:13-21, 1994; see also U.S. Pat. Nos. 5,545,806 and 5,569,825, both of 
which are hereby incorporated by reference). 
The methods described herein may be performed, for example, by utilizing 
pre-packaged kits comprising at least one specific UCH nucleotide sequence 
or antibody reagent described herein, which may be conveniently used, for 
example, in clinical settings, to diagnose and treat conditions associated 
with an increased (or decreased) resistance to chemical agents. 
The activity of UCH can be decreased with chemical compounds such as sodium 
borohydrate, and sulfhydryl blocking agents such as iodoacetamide. 
However, it is more likely that small molecules, for example those 
identified by the screening methods described herein, will be clinically 
useful. 
Increasing the Resistance of a Cell to a Chemical Agent 
The resistance of a cell to a chemical agent can be increased (i.e., the 
cell can be made less sensitive to the effects of the agent), by 
increasing the expression or activity of UCH. The effects of the chemical 
agent can be, but are not necessarily, deleterious to the cell. Chemical 
agents that can be used in the practice of the invention are described 
more fully below. A cell will be made more resistant to a chemical agent 
if it continues to perform one or more of the physiological functions that 
a cell of that type normally performs, or if it survives longer in the 
presence of the chemical agent that is cytotoxic than does a cell whose 
resistance has not been increased. 
Skilled artisans are readily able to determine the circumstances in which 
it would be desirable to increase the resistance of a cell to a 
deleterious chemical agent. Generally, these circumstances include any 
situation in which it is desirable to protect a cell from a chemical 
agent. For example, it could be desirable to protect certain plants in the 
vicinity of others that are being treated with an herbicide. Similarly, it 
could be desirable to protect healthy cells within an animal that are 
exposed to a chemotherapeutic agent that is administered to treat 
cancerous cells within the same animal. 
The expression of UCH can be increased within a cell in numerous ways, 
including those described below. 
Increasing the Expression of UCH by Transfection with UCH-encoding Nucleic 
Acid Sequences 
The expression of UCH within a cell can be increased by transfecting the 
cell with a nucleic acid molecule that contains a sequence encoding UCH. 
The sequences encoding many forms of UCH are known, and widely available. 
These sequences can be ligated into expression vectors and transfected 
into cells using no more than routine, molecular biological techniques. 
For example, a nucleic acid sequence encoding UCH can be ligated into a 
plasmid such as the pMSXND expression vector, which is commonly used to 
transfect mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 
1988). 
The nucleic acid molecules inserted into the vector may encode a 
full-length UCH polypeptide, or a biologically active fragment thereof. A 
biologically active fragment of UCH would be a fragment that can cleave 
the bond between ubiquitin and a second protein that has been targeted for 
degradation. Preferably, the encoded polypeptide fragment will exhibit at 
least 50%, more preferably at least 70%, and most preferably at least 90% 
(e.g., 95% or even 99%) of the activity of the full-length UCH enzyme of 
which it was a part. The sequence encoding a biologically active fragment 
of UCH fragment can, if desired, be obtained by amplifying a region of the 
full-length UCH nucleic acid molecule by the polymerase chain reaction 
(PCR), or generated by treating the full-length UCH nucleic acid molecule 
with one or more restriction endonucleases. 
Nucleic acid molecules useful in the practice of the invention can contain 
either naturally occurring sequences, or sequences that differ from those 
that occur naturally, but, due to the degeneracy of the genetic code, 
encode UCH. 
The nucleic acid molecule that is inserted into an expression vector can be 
operably linked to a regulatory sequence, such as that of a promoter or 
enhancer, which will enhance the expression of the inserted nucleic acid 
molecule. These regulatory sequences can be those that are naturally 
associated with genes encoding UCH, i.e., they can include some or all of 
the non-coding nucleic acid sequence that lies upstream or downstream from 
a UCH coding sequence in the naturally occurring genome of a eukaryotic 
cell. Alternatively, the regulatory elements included in the expression 
vector can be heterologous elements. Cytomegalovirus or metallothionein 
promoters are commonly used in enhance expression in mammalian cells. In 
the case of higher eukaryotes, tissue-specific and cell type-specific 
promoters are also widely available. These promoters are so named for 
their ability to direct expression of a nucleic acid molecule in a given 
tissue or cell type that can be maintained in tissue culture or that can 
be present within the body. Skilled artisans are well aware of numerous 
promoters and other regulatory elements which can be used to direct 
expression of nucleic acid molecules. 
The recombinant technology required to clone and express such genes is 
standard in the art and is described in detail, for example, in Sambrook 
et al. (Molecular Cloning: A Laboratory Manual, CSH Laboratory Press, Cold 
Spring Harbor, N.Y., 1989). 
In addition to sequences that facilitate transcription of the inserted 
nucleic acid molecule, vectors can contain origins of replication, and 
other genes that encode a selectable or detectable marker. For example, 
the neomycin-resistance (neo.sup.r) gene imparts G418 resistance to cells 
in which it is expressed, and thus permits phenotypic selection of the 
transfected cells. Examples of marker or reporter genes include 
.beta.-lactamase, chloramphenicol acetyltransferase (CAT), adenosine 
deaminase (ADA), aminoglycoside phosphotransferase (neo.sup.r, G418.sup.r) 
dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), 
thymidine kinase (TK), lacZ (encoding .beta.-galactosidase), and xanthine 
guanine phosphoribosyltransferase (XGPRT). Those of ordinary skill in the 
art can readily determine whether a given regulatory element or selectable 
marker is suitable for use in a particular experimental context. 
Furthermore, and as with many of the standard procedures associated with 
the practice of the invention, skilled artisans will be aware of 
additional useful reagents, for example, of additional sequences that can 
serve the function of a marker or reporter. 
Viral vectors can also be used in the invention and include, for example, 
retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian 
virus 40 (SV40), and bovine papilloma virus vectors (see, for example, 
Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring 
Harbor, N.Y.). If additional guidance is required in creating an 
expression vector, skilled artisans may consult Ausubel et al. (Current 
Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1993) 
and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 
1987). 
The nucleic acid molecules of the invention can be synthesized (e.g., by 
phosphoramidite-based synthesis) or obtained from a biological cell, such 
as a fungal, plant, or animal cell. Combinations or modifications of the 
nucleotides within these cell types are also encompassed. 
The vectors described above can be used to obtain a transient change in the 
expression of UCH within a biological cell, or they can be used to 
incorporate a nucleic acid molecule encoding UCH into the genome of a 
heterologous cell (or into the genome of a homologous cell, at a position 
other than the natural chromosomal location). 
Those of ordinary skill in the art will understand that there are numerous 
ways of transfecting biological cells with the nucleic acid molecules 
described above. For example, cells can be transfected with plasmid 
vectors by standard methods including, but not limited to, 
liposome-polybrene-, or DEAE dextran-mediated transfection (see, e.g., 
Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., 
Neurosci. Lett. 117:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 
1989), electroporation (Neumann et al., EMBO J. 7:841, 1980), calcium 
phosphate precipitation (Graham et al., Virology 52:456, 1973; Wigler et 
al., Cell 14:725, 1978; Felgner et al., supra) microinjection (Wolff et 
al., Science 247:1465, 1990), or velocity driven microprojectiles 
("biolistics"). 
Effective Dose 
Toxicity and therapeutic efficacy of a given compound (e.g., UCH or a 
compound that can alter the level of expression or activity of UCH) can be 
determined by standard pharmaceutical procedures, using either cells in 
culture or experimental animals to determine the LD.sub.50 (the dose 
lethal to 50% of the population) and the ED.sub.50 (the dose 
therapeutically effective in 50% of the population). The dose ratio 
between toxic and therapeutic effects is the therapeutic index and it can 
be expressed as the ratio LD.sub.50 /ED.sub.50. Compounds which exhibit 
large therapeutic indices are preferred. While compounds that exhibit 
toxic side effects may be used, care should be taken to design a delivery 
system that targets such compounds to the site of affected tissue in order 
to minimize potential damage to unaffected cells and, thereby, reduce side 
effects. 
Data obtained from the cell culture assays and animal studies can be used 
in formulating a range of dosage for use in humans. The dosage of such 
compounds lies preferably within a range of circulating concentrations 
that include the ED.sub.50 with little or no toxicity. The dosage may vary 
within this range depending upon the dosage form employed and the route of 
administration utilized. For any compound used in the method of the 
invention, the therapeutically effective dose can be estimated initially 
from cell culture assays. A dose may be formulated in animal models to 
achieve a circulating plasma concentration range that includes the 
IC.sub.50 (that is, the concentration of the test compound which achieves 
a half-maximal inhibition of symptoms) as determined in cell culture. Such 
information can be used to more accurately determine useful doses in 
humans. Levels in plasma may be measured, for example, by high performance 
liquid chromatography. 
Formulations for Use and Routes of Administration 
In therapeutic applications, UCH, or compounds that alter its expression or 
activity, can be administered with a physiologically-acceptable carrier, 
such as physiological saline. The therapeutic compositions of the 
invention can also contain a carrier or excipient, many of which are known 
to skilled artisans. Excipients which can be used include buffers (for 
example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate 
buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, 
proteins (for example, serum albumin), EDTA, sodium chloride, liposomes, 
mannitol, sorbitol, and glycerol. The polypeptides of the invention can be 
formulated in various ways, according to the corresponding route of 
administration. For example, liquid solutions can be made for ingestion or 
injection; gels or powders can be made for ingestion, inhalation, or 
topical application. Methods for making such formulations are well known 
and can be found in, for example, "Remington's Pharmaceutical Sciences. 
Routes of administration are also well known to skilled pharmacologists and 
physicians and include intraperitoneal, intramuscular, subcutaneous, and 
intravenous administration. Additional routes include intracranial (e.g., 
intracisternal or intraventricular), intraorbital, opthalmic, 
intracapsular, intraspinal, intraperitoneal, transmucosal, topical, 
subcutaneous, and oral administration. It is expected that the intravenous 
route will be preferred for the administration of UCH. The subcutaneous 
route may also be used frequently as the subcutaneous tissue provides a 
stable environment for the polypeptide, from which it can be slowly 
released. 
UCH polypeptides, administered according to the methods of the invention, 
are less likely to be immunogenic than many therapeutic polypeptides 
because they can be identical to a wild-type polypeptides, i.e., UCH 
enzymes, within the cell. 
It is well known in the medical arts that dosages for any one patient 
depend on many factors, including the general health, sex, weight, body 
surface area, and age of the patient, as well as the particular compound 
to be administered, the time and route of administration, and other drugs 
being administered concurrently. Dosages for the polypeptide of the 
invention will vary, but can, when administered intravenously, be given in 
doses of approximately 0.01 mg to 100 mg/ml blood volume. A dosage can be 
administered one or more times per day, if necessary, and treatment can be 
continued for prolonged periods of time, up to and including the lifetime 
of the patient being treated. If a polypeptide of the invention is 
administered subcutaneously, the dosage can be reduced, and/or the 
polypeptide can be administered less frequently. Determination of a 
correct dosage for a given application is well within the abilities of one 
of ordinary skill in the art of pharmacology. 
Methods for Screening Cytotoxic Agents 
Altering the resistance of a cell to cytotoxic agents, by modulating the 
level of UCH expression or activity, creates a unique population of cells 
that can be used to screen cytotoxic agents. Cells that have been treated 
with an agent that increases the expression or activity of UCH, and are 
therefore more resistant to cytotoxic agents, can be used to identify 
especially potent cytotoxic agents (i.e., cytotoxins that can kill or 
otherwise harm cells that have a high level of UCH expression or 
activity). These agents could be useful in treating cancers that are 
resistant to presently used chemotherapeutics. A screening method 
developed on this premise could be carried out as follows. A cell that has 
been treated with an agent that increases the expression or activity of 
UCH is contacted with a candidate cytotoxic agent, and the survival of the 
cell is assessed; a decrease in cell survival would indicate that the 
candidate cytotoxic agent is a potent cytotoxic agent. 
Similarly, weak cytotoxic agents, that could be overlooked in currently 
available screens, can be identified using cells that have been treated 
with an agent that decreases UCH expression or activity, and thereby 
renders the cell more sensitive to cytotoxic agents. Compounds could be 
screened in this context as follows. A cell that has been treated with an 
agent that decreases the expression or activity of UCH is contacted with a 
candidate cytotoxic agent, and the survival of the cell is assessed; a 
decrease in cell survival indicating the presence of a candidate cytotoxic 
agent the would have remained undetectable had the cell not been rendered 
more sensitive to its action (e.g., if the cell had not been treated with 
an agent that decreases the expression or activity of UCH). Although 
discovered as a weak cytotoxic agent, those of skill in the art of 
pharmacology are aware of modifications that could increase the potency of 
cytotoxic agents discovered according to this method. 
Identification of Compounds that Modulate the Expression or Activity of UCH 
Also featured in the invention are methods of identifying compounds that 
increase or decrease the expression or activity of UCH in vivo. For 
example, to discover such compounds, cells that express UCH are cultured, 
exposed to a test compound (or a mixture of test compounds), and the level 
of UCH expression or activity is compared with the level of expression or 
activity in cells that are otherwise identical but that have not been 
exposed to the test compound(s). Many standard quantitative assays of gene 
expression, which are known to those of skill in the art, can be utilized 
in this aspect of the invention. Virtually any compound can be tested, 
including small molecules, polypeptides (e.g., oligopeptides, antibodies, 
or antibody fragments), and nucleic acid molecules. Compounds identified 
in this way can be used as agents to modulate UCH expression in vivo and, 
thereby alter the resistance of the treated cell to cytotoxic agents 
(including, but not limited to doxorubicin, vincristine, colchicine, 
VP-16, vinblastine, verapamil, mitoxantrone, taxol, Cyclosporin A, 
quinidine, progesterone, tamoxifen, epirubicin, daunorubicin, MX2, and 
heavy metal ions such as arsentie, arsenate, antimony tartrate, animonate, 
and cadmium, whether alone or in any combination). 
Following is an example, which is provided for the purpose of illustrating, 
not limiting, the invention. 
EXAMPLE 
UCH Expression is Upregulated in Drug-Resistant Cells 
Expression of UCTH-L1 was examined in drug-sensitive and drug-resistant 
UCLA-P3, A2780, and MCF-7 cancer cells by Northern blot analysis, as 
follows. 
Preparation of Drug-Resistant Cells 
UCLA-P3 cells were derived from a human non-small cell lung carcinoma. A 
drug resistant variant, UCLA-P3-0.003VLB cells, was selected in vitro for 
resistance to 3 ng/ml desacetylvinblastine hemisuccinate over a ten month 
period of time. Both wild-type and drug resistant cells were cultured in 
RPMI 1640 containing L-glutamine and 25 mM HEPES buffer (Gibco BRL), 
supplemented with 10% fetal bovine serum (FBS) and 50 mg/ml gentamycin. 
The cells were passaged twice weekly by splitting them in ratios of 1:6 or 
1:8. 
A2780 cells were derived from a human ovarian carcinoma. A doxorubicin 
resistant variant, 2780AD, was developed by maintaining the A2780 cells in 
culture with 2 mM doxorubicin (Hamilton et al., Semin. Oncol. 11:285-298, 
1984). Both the drug-sensitive and drug-resistant cells can be cultured in 
RPMI medium, as described above, that contains additional L-glutamine (to 
a final concentration of 2 mM) and 10 .mu.g/ml bovine insulin. 
MCF-7 cells are derived from a human breast carcinoma. Doxorubicin (also 
known as adriamycin) was used to generate a line of MCF-7 cells that 
exhibit cross-resistance to a wide range of anti-cancer drugs. This cell 
line was established by Batist et al. (J. Biol. Chem. 261:15544-15549, 
1986), as follows. Adriamycin-resistant MCF-7 cells were selected by 
culturing non-resistant cells in gradually increasing concentrations of 
adriamycin, with the initial concentration of adriamycin being 10.sup.-8 
M. When the cells were able to survive at any given concentration of drug, 
they were passaged in concentrations that were 1.5-fold to 2-fold higher. 
The cells finally obtained were able to survive in 10.sup.-5 M adriamycin. 
In preparation for the experiments described below, drug-sensitive and 
drug-resistant MCF-7 cells were cultured in RPMI 1640 medium containing 
L-glutamine, 25 mM HEPES buffer, 10% FBS, and 50 .mu.g/ml gentamycin. 
RNA Extraction and Analysis of UCH Expression 
The cell lines described above (i.e., drug-sensitive and drug-resistant 
MCF-7, A2780, and UCLA-P3 cells) were cultured until confluent, and the 
culture medium was removed by aspiration and replaced with RLT buffer 
containing .beta.-mercaptoethanol (1 .mu.l .beta.-mercaptoethanol/100 
.mu.l RLT buffer; Qiagen), which lyses the cells and stabilizes RNA. The 
lysates were harvested and homogenized by spinning through a Quiashredder 
column (Qiagen), and the RNA was analyzed by Northern blot. 
As shown in FIG. 2, both drug-reistant MCF-7 cells and drug-resistant A2780 
cells showed an upregulation in UCTH-L1 expression compared to their 
drug-sensitive counterparts. Drug-sensitive A2780 cells exhibit a basal 
level expression of UCTH-L1 whereas drug-sensitive MCF-7 cells appear to 
have little or no UCTH-L1 expression. Both drug-resistant and 
drug-sensitive UCLA-P3 cells failed to show expression of UCTH-L1 message 
by Northern blot.