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`Volume 13 - Number 6 - 19 September 1996
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`HEALTH SCIEMEES LIBRARY
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`

`

`Taxol resistance of p185-overexpressing breast cancer cells
`D Yu et al
`
`for
`expression may be a useful prognostic factor
`predicting responsiveness of breast cancers
`to che-
`motherapy.
`In this study, we investigated whether overexpression
`of p185 may lead to increased drug resistance in
`human breast cancers by comparing p185-low-expres—
`sing MDA—MB—435 parental
`cells with the p185-
`overexpressing MDA—MB-435 c-erbB—2 stable transfec-
`tants
`for Taxol
`sensitivity. We
`also investigated
`iwhether cell cycle characteristics, oncogenic transfor-
`mation, and the ma’r-Z-encoded p170 may be involved
`in the increased Taxol resistance.
`
`Results
`
`Increased Taxol resistance in MDA—MB-435 c—erbB—Z
`
`transféctcmts
`
`To examine whether c-erbB-Z/neu overexpression can
`lead to increased chemoresistance in breast cancer cells,
`we needed to have a panel of human breast cancer cell
`lines with a similar genetic background but expressing
`different levels of p185. Differences in chemosensitivity
`among these cells should be caused by the differences
`in p185 expression. To achieve this, we used the MDA-
`MB-435 human breast cancer cells that express very
`low levels of p185 and are highly sensitive to Taxol as
`the recipient
`for transfection. We cotransfected the
`pCMVerbB-2 plasmid containing the 4.4 kb full—length
`human c-erbB—Z cDNA (Yamamoto et (11., 1986) and
`the pSV2-neo plasmid carrying the neomycin-resistance
`selection marker gene into MDA—MB—435 cells. G418-
`resistant clones were selected and expanded into cell
`lines that were designated 435.eB cell lines. The same
`approach was used to select control 435.neo and
`435.neo.pool cell lines, in which the pSV2-neo plasmid
`alone was transfected into MDA-MB-435 cells, and
`individual G418-resistant clone (435.neo) or pooled
`G418-resistant clones
`(435.neo.pool) were expanded
`into cell
`lines. It is possible that some of the 435.eB
`stable transfectants selected by such a cotransfection
`strategy may only harbor the neomycin—resistance gene
`but not the c-erbB-2 gene. Therefore, to identify those
`435.eB transfectants that integrated the transfected c-
`erbB-Z gene and actually produced p185 protein,
`immunoblot analysis with c-neu—Ab-3 antibodies were
`performed (Figure 1a). Four of the 435.eB transfec-
`tants
`(435.eB1,
`435.eB2,
`435.eB3
`and
`435.eB4)
`expressed increased amounts of p185 protein com-
`pared with the parental MDA-MB-435 cells. The
`control
`435.neo
`and
`435.neo.pool
`cell
`lines,
`as
`expected, did not express increased amounts of p185
`protein. The relative amounts of p185 in the 435.neo
`and 435.neo.pool
`cells and the 435.eBl, 435.eB2,
`435.eB3 and 435.eB4 transfectants are 1.16-, 0.99-,
`258-, 149-, 135- and l65-fold of that of the parental
`MDA-MB—435 cells
`(Table
`l),
`as determined by
`quantitation with the Personal Densitometor 50371
`(Molecular Dynamics, Sunnyvale, CA). Equal loading
`of protein lysates were confirmed by Western blot on
`oc-actin using anti-oc-actin monoclonal antibodies (Ab-l,
`Oncogene Science Inc., data not shown). It is notable
`that
`the p185 level
`in the 435.eB transfectant
`is
`comparable to that
`in the SK-BR-3 breast cancer
`cells established from a different patient, which express
`
`
`
`a
`
`m
`g
`<
`D
`E
`
`F-
`O
`t:
`a:
`
`<r
`a.)
`m
`
`m
`E
`m
`U)
`>4
`
`9185—
`
`n.u .
`
`b
`
`r—
`o
`a:
`C
`
`DC
`“.
`no
`a:
`
`p185—
`
`‘1.
`to
`(D
`
`EC
`“1
`no
`(D
`
`a.
`
`(a) Immunoblot analysis for the e—erbB-Z/neu—encoded
`Figure 1
`p185 proteins in the cell lysates of the indicated cell lines. 100 lig
`of protein from each sample was electrophoresed on 6% SDS—
`PAGE and transferred
`to nitrocellulose. The
`filters were
`incubated with the primary antibody c-neu—Ab—3 against p185.
`Position of the p185 is indicated on the left. The amounts of p185
`in the MDA«MB—435 cells, 435.ne0 and 435.neo.pool cells and the
`435.eBl, 435.eB2, 435.eB3, 435.eB4 transfectants were determined
`by quantitation with the Personal Densitometor 50371 (Molecular
`Dynamics, Sunnyvale, CA)
`and
`shown in Table
`l.
`(b)
`Immunoblot analysis for the e-erbB-Z/neu-encoded p185 proteins
`in the cell
`lysates of the 435.neo.1, eB.lR, eB.2R, and 435.eB4
`cells.
`Immunoblot analysis were performed as
`in (3). Equal
`loading of protein lysates was confirmed by Western blot on or-
`actin using Ab-l
`anti—oc—actin monoclonal antibodies (data not
`shown)
`
`Table 1 Characteristics of cell lines
`
` Doubling time S—phase fractim
`
`Cell lines
`play-“32
`(h)
`(%)
`MDA-MB-435
`1.00
`20.53
`49.70
`435.neo.1
`1.16
`20.01
`37.70
`435.neo.pool
`0.99
`18.43
`45.00
`435.eB.1
`258.00
`17.94
`38.90
`435.eB.2
`149.00
`16.62
`36.40
`435.eB.3
`135.00
`18.11
`41.35
`
`435.eB.4 40.50 165.00 18.92
`
`
`
`Data represent an average of at least two different experiments. SDs
`for each point were less than 10%
`
`l28—fold (Kraus er (1]., 1987) and
`c-erbB-Z mRNA at
`p185 at 262—fold of that of the MDA—MB-435 cells
`(Figure la). We thus established a panel of stable
`transfectants that express differently increased amounts
`of p185 which are parallel
`to the c-erbB—2 gmc
`expression levels in primary human breast cancers.
`The 435.neo and 435.neo.pool cells were used as
`control cell
`lines to make sure that
`the changes in
`biological properties,
`such as
`chemosensitivity t0
`Taxol, in the 435.eB transfectants were not caused by
`the selection process or by transfection of the plasmids
`or the expression of pSV2-neo gene.
`Once we established the MDA-MB-435 transfec-
`
`tants, we compared the Taxol chemosensitivity of the
`plSS-overexpressing transfectants (435.eB lines)
`\T‘ith
`that of
`the
`p185-low—expressing
`control
`435.1160:
`435.neo.pool, and parental MDA-MB-435 cells using
`
`MDA435 transfectants
`_
`8
`q‘
`O
`C
`a)
`
`‘—
`a:
`m
`
`N
`-
`a:
`m
`
`a;
`m
`
`the clonogenic assays (Cree et al., 1995). Cells were
`
`

`

`Taxol resistance of p185-overexpressing breast cancer cells
`D Yu et al
`
`1361
`
`(Figure 2a). The
`and parental MDA-MB—435 cells
`difference in Taxol
`sensitivity between the 435.eB
`transfectants and the control 435.neo, 435.neo.pool
`and parental MDA—MB—435 cells were statistically
`highly significant
`(P<0.00012). We also compared
`the Taxol sensitivities of the 435.eB transfectants and
`
`the control cell lines using the CellTiter96TM AQ Non-
`Radioactive Cell Proliferation Assay (Promega Corp)
`(Promega, 1991). The Taxol IC50 values of the p185-
`overexpressing 435.eB transfectants from three inde-
`pendent experiments were significantly higher than that
`of the control 435.neo, 435.neo.pool and the parental
`MDA-MB—435 cells (P<0.01), ranging from a 4.2— to
`9-fold
`increase
`(data not
`shown). These
`results
`demonstrate that overexpression of p185 can lead to
`increased resistance to Taxol in breast cancer cells.
`
`Interestingly, several spontaneous revertants of the
`435.eB transfectants appeared after ~25 passages that
`lost the transfected c—erbB—Z gene and express very low
`levels of p185 protein (Figure 1b, eB.1R named for
`revertants derived from the 435.eB1 transfectants and
`eB.2R for revertants derived from the 435.eB.2 transfec-
`
`tants). When we tested the Taxol chemosensitivity of the
`plSS-low-expressing spontaneous revertants, these rever-
`tants demonstrated similar Taxol sensitivity as the control
`435.neo.1 cells,
`i.e.,
`these revertants have lost Taxol
`resistance (Figure 213). These data demonstrate that the
`increased Taxol resistance in the 435.eB transfectants was
`
`indeed the result of p185 overexpression.
`To examine whether increased Taxol resistance of the
`
`435.eB transfectants was associated with changes in cell
`proliferation characteristics, we measured the doubling
`times and S-phase fractions of the pl85-overexpressing
`435.eB transfectants and the plSS-low—expressing con—
`trol cells. No statistically significant difference was
`observed among the Taxol-resistant 435.eB transfec-
`tants and the Taxol-sensitive control cells (Table 1).
`These
`findings
`indicate
`that
`the
`increased Taxol
`resistance of the 435.eB transfectants did not result
`
`from any differences in growth rate and DNA synthesis
`rate but was the result of p185 overexpression.
`been
`Since overexpression of
`c-erbB-Z/neu has
`reported to induce transformation in NIH3T3 cells
`(Hudziak et al., 1987), we investigated whether the
`increased Taxol resistance is associated with oncogenic
`transformation. The neu-transformed NIH3T3 cells
`
`(EJ-ras),
`res—transformed NIH3T3 cells
`(B104-1-1),
`and the parental nontransformed NIH3T3 cells were
`examined for their sensitivity to Taxol (Figure 2c). The
`parental NIH3T3 cells were sensitive to Taxol. The
`neu—transformed B104-1-1 cells were highly resistant to
`Taxol, which is consistent with our observations in
`p185-overexpressing 435.eB transfectants. However, the
`ras-transformed EJ-ras cells were sensitive to Taxol as
`
`the parental NIH3T3 cells. These results indicate that
`the increased resistance to Taxol in lieu-transformed
`
`NIH3T3 cells and p185-overexpressing breast cancer
`cells is associated with c—erbB—Z/neu gene expression
`but not a general effect of oncogenic transformation.
`
`Overexpression 0fp185 can confer intrinsic Taxol
`resistance via mdr-l independent mechanisms
`
`is a
`The mdr-I gene-encoded P-glycoprotein (p170)
`transmembrane transport protein that functions as an
`ATP-dependent drug-efllux pump, reduces drug accu-
`
`
`
`grown in the presence of different concentrations of
`Taxol, and the percentages of viable cell clones relative
`to the drug-free control were quantitated. The 435.eB
`transfectants demonstrated increased resistance
`to
`
`Taxol killing than the control 435.neo, 435.neo.pool,
`
`MDA-MB—435
`
`435mm
`
`435.neo.pool
`
`435.1231
`
`435.5132
`435.eB.3
`
`435.cB.4
`
`0.0001
`
`0.001
`
`0.01
`
`0.1
`
`Taxol Concentration (pg/ml)
`
`'5
`>"-1
`
`2
`:1
`CD
`
`U S
`
`-1
`N
`.:
`:3
`t
`U)
`
`B F
`
`b
`
`0
`
`>1
`
`
`
`""u""
`
`435.neo
`
`—o— 435.eB.l.R
`
`—o— 435.eB.2.R
`
`‘
`
`.
`“D
`1‘
`
`‘
`
`0.01
`
`0.1
`
`1
`
`10
`
`100
`
`Taxol Concentration (11M)
`
`NIH3T3
`
`Elm
`
`—0— 1310411
`
`100
`
`75
`
`50
`
`25
`
`0 0
`
`.001
`
`100
`
`_ 75
`C“
`>I—
`.1
`:1
`U:
`
`50
`
`0.01
`
`Oil
`
`1
`
`IO
`
`100
`
`5°
`
`25
`
`00
`
`.001
`
`Taxol Concentration (uM)
`
`(21) p185 overexpressing 435.eB transfectants are more
`Figure 2
`to Taxol
`than the MDA-MB-435,
`435.neo,
`and
`resistant
`435.neo.pool cells that express basal level of p185. Cytotoxicities
`of Taxol
`in the indicated MDA—MB-435 cells or transfectants
`were determined by clonogenic assays and were shown as Taxol
`dose-response curves of the indicated cell lines. The means of at
`least
`three independent experiments were plotted.
`(b) Similar
`Taxol sensitivities among the 435.neo.l control cells,
`the eBilR
`and eB.2R revertants. Taxol dose-response curves were deter-
`mined by using the CellTiter96TM AQ Non-Radioactive Cell
`Proliferation Assay (Promega Corp) and were averaged from
`three independent experiments. (c) Increased Taxol resistance in
`new—transformed NIH3T3 cells (BlO4-l-1) but not
`in parental
`NIH3T3 cells nor
`in Ins-transformed NIH3T3 cells
`(EJ—ras).
`Taxol dose-response curves were determined as in (b) and were
`averaged from three independent experiments
`
`
`
`

`

`1362
`
`Taxol resistance of p185-overexpressing breast cancer cells
`D Yu et al
`
`MDA435 transfectants
`
`m
`a
`<
`0
`2
`
`_
`8
`Q.-
`°
`and
`
`“.
`°
`GED
`
`cg
`
`m
`m
`
`2
`(D
`
`2
`a)
`
`m
`I
`2'2
`(I)
`
`13170 fi~~—’“n‘-- .
`
`Fibronectin ‘—' -— ;- ~—-— *- “‘
`
`"—
`
`”'
`
`p170|eve|
`
`1.0
`
`0.9
`
`1.1
`
`0.9 1.10.8 1.2
`
`12.2
`
`the mdr—I—encoded
`Immunoblot analysis for
`(Top):
`Figure 3
`p170 proteins in the cell lysates of the indicated cell lines. 100 11g
`of protein from each sample was electrophoresed on 6% SDS~
`PAGE and transferred to nitrocellulose filters. The filters were
`incubated with the primary polyclonal antibody against p170
`(Oncogene Sci. 1110.). Position of the p170 is indicated on the left.
`(Middle): Immunoblot analysis for fibronectin as internal loading
`controls. The p170 signals on the same filter used in Figure 3 (a)
`were stripped away and the filter was re—hybridized with anti-
`fibroncctin antibodies (Oncogene Science, Inc). Position of the
`fibronectin protein is
`indicated on the left. The p170 and
`fibronectin protein levels were quantified by personal
`laser
`densitometor. The
`relative amount of mdr—J—encoded p170
`expression levels in each cell
`line was normalized by fibronectin
`level
`in the corresponding cell
`line and were indicated in panel
`(Bottom)
`
`resistance to a
`mulation, and can therefore confer
`(Moscow and
`variety of chemotherapeutic drugs
`Cowan,'l988; Nooter and Herweijer, 1991). Recently,
`a statistically significant association has been reported
`between the expression of the c-erbB-Z/neu-encoded
`p185 and the mdr—I-encoded p170 in mammary
`carcinomas
`(Schneider
`cl
`(1].,
`1994). We therefore
`investigated whether mdr-I may
`be
`involved
`in
`increased Taxol
`resistance of p185 overexpressing
`breast
`cancer
`cells. We performed Western blot
`analysis on the MDA—MB—435, 435.neo, 435.neo.pool
`cells and the 435.eB transfectants using the polyclonal
`anti-human P-glycoprotein antibody raised against the
`P-glycoprotein C-terminal cytoplasmic domain (Onco—
`gene Science, Inc.) (Figure 3, top). A very low level of
`the mdr-I-encoded P-glycoprotein was detected in the
`MDA-MB—435 cells and the levels of
`the mdr-I-
`
`pl85—overexpressing 435.eB
`the
`in
`encoded p170
`similar
`to
`the MDA-MB—435,
`transfectants were
`435.neo, and 435.neo.pool cells that expressed basal
`levels of p185. Western blot analysis on the panel of
`transfectants were also performed using anti-fibronec-
`tin monoclonal antibodies (Oncogene Science, Inc.) as
`internal
`loading control
`(Figure 3, middle) and the
`p170 and fibronectin protein levels were quantified by
`laser densitometor. The mdr-I-encoded p170 expression
`levels
`in
`the
`transfectants were
`normalized
`by
`fibronectin levels for quantitative comparison which
`demonstrated that
`the relative amount of mdr-I—
`
`encoded p170 are very similar among the C—erbB—2-
`transfectants and control cells (Figure 3, bottom). The
`results indicated that c-erbB-Z did not up~regulate mdr-
`]~encoded p170 expression in 435.eB transfectants,
`suggesting that mdr-I may not be involved in increased
`Taxol
`resistance of the p185—overexpressing 435.cB
`transfectants.
`
`
`
`in
`the involvement of mdr—I
`To further rule out
`Taxol
`resistance of 435.eB transfectants, we used
`thioradazine, a chemosensitizing agent or multidrug—
`resistance reversing agent that can block P-glycoprotein
`function, on these 435.cB transfectants. We evaluated
`whether these 435.eB transfectants can be sensitized to
`
`Taxol by the mdr—J blocker treatment. Non-cytotoxic
`dose of thioridazine (1 rig/ml) has been shown to
`effectively block p170 function and restore drug
`sensitivity for multidrug—resistant sarcoma 8180 cell
`by binding to P-glycoprotein (Efferth and Volm, 1993).
`Therefore 1—2 rig/ml of thioridazine, which did no‘
`yield significant cytotoxicity on MDA-MB-435 cells
`(< 10% reduction of cell survival), were tested for tht
`Taxol sensitization effect. Compared to Taxol alone
`thioradazine plus Taxol did not
`lead to remarkable
`increase of Taxol sensitivity in either the pl85-lou
`expressing MDA-MB-435 cells or the p185-overexpres-
`sing 435.eB2 and 435.eB4 transfectants (Figure 4a,
`.-
`and d). This phenomenon was also observed over a
`range of Taxol doses (0.02, 0.05, 0.1, 0.2 and 0.5 nM)
`and on other cell
`lines, where thioridazine had ro
`differential sensitization effect on 435.neo, 435.neo.pool
`cells and other 435.eB transfectants (data not show).
`In contrast, 172 ug/ml thioridazine had striking Taxol
`sensitization effect on the CHO.VBR1.5-l cells (Figu.e
`4b),
`that has
`been reported to be
`resistant
`to
`vinblastine due
`to overexpression of mdr-encoded
`p170 (Kuo er (1]., 1994). Taken together, lack of Ta: )1
`sensitization by thioridazine on the Taxol-resistant
`435.eB transfectants was due to the low levels of
`
`p170 expression and function in these cells. Therefore,
`mdr-I is not involved in the increased Taxol resistance
`of the 435.eB transfectants.
`
`tumors of pet-“ems
`
`Discussion
`
`p185
`the C—erbB—Z/neu-encoded
`Overexpression of
`protein has been found in many types of human
`cancers and has been correlated with poor clinical
`outcome in breast and ovarian cancers (Hung, 1968;
`Slamon e!
`(1]., 1989). Here, we demonstrated “‘at
`overexpression of p185 can confer resistance to Taxol
`in human breast cancer cell
`lines.
`It
`is notable .iiat
`Taxol has attracted intense interest because of its
`antitumor activity in
`drug—refractory ovarian and
`breast
`carcinomas. However,
`about
`30—40% Of
`patients do not respond to Taxol
`treatment (Cheva:
`lier er (1]., 1995; Holmes er 0]., 1991). Overexpressitn Of
`the c-erbB—2/neu gene has been found in about 20-
`30% of breast cancers and our results indicate that
`breast cancers that overexpress p185 will not respond
`well to Taxol. Interestingly, we have examined 'i’axol
`sensitivities of seven breast cancer cells lines establ’llt’fd
`from different patients using clonogenic assays and
`found that those c-erbB-Z overexpressing breast t. .1CCI‘
`cell lines are significantly more resistant to Taxol than
`those c-erbB-Z low~expressing breast cancer cell
`lines
`(data not shown). These findings may provide one
`explanation on why some breast cancer patients (10 ml
`respond to Taxol
`treatment. This important
`is
`16
`15
`currently being further pursued by examining whether
`overexpression of p185
`correlates with
`increased
`chemoresistance in primary breast
`
`

`

`
`
`(1].,
`trials (Holmes er
`enrolled in the Taxol Clinical
`1991). Nevertheless, our results agree with a previous
`clinical study where p185 overexpressing breast tumors
`were
`found
`less
`responsive
`to CMF-containing
`
`than those with a normal
`chemotherapy regimens
`amount of the gene product (Gusterson ez‘
`(11., 1992).
`Therefore, p185 expression level may be a potential
`predictive factor that can be used to select patients who
`
`Taxol resistance of p185-overexpressing breast cancer cells
`D Yu et aI
`1363
`
`MDA-MB-435
`
`CHO.VBR1.5-1
`
`a Thioridazine, 0
`
`[25
`
`Thioridazine.0
`
`.
`.
`.
`El ThioridazmeJug/ml
`
`7
`I
`‘
`E Th‘
`iorida21nc,kug/ml
`
`
`
`xxx
`
`(t
`x:\
`3.)
`\(x
`xxx
`\(x
`\I\
`:3:
`\
`«I
`\:‘\
`\ Ix
`\(\.
`‘\:\
`K!\
`“A. N
`
`
`
`
`
`
`_
`N
`.>
`;
`h
`CE
`Q
`§
`
`100
`
`75
`
`50
`
`25
`
`0
`
`E3 Thioridazinefipglml
`
`El
`
`Thioridazine, 211g/ml
`
`
`:\:\
`Ix!»
`xx"
`l“)
`xx)
`(xix
`N \
`(xxx
`3;
`x \.
`:9
`’\"\
`Ixfx
`’\l\
`’\,\
`’\’\
`/\/N
`
`
`
`x I
`‘s
`'K
`(\l‘s
`3‘;
`'\ \
`jg
`"sx‘.
`[\J\
`"u/‘s
`’\x\
`"x/‘a.
`I’\('\
`
`O
`0.05
`TaxolmM)
`
`0
`
`5
`Tax01(HM)
`
`10
`
`435_eB,2
`
`435.eB.4
`
`d
`
`7“
`I:
`>
`L:
`5
`CI)
`g
`
`125
`
`'00
`
`75
`
`50
`
`25
`
`0
`
`c
`
`%Survival
`
`125
`
`l ()0
`
`75
`
`50
`
`25
`
`125
`
`l ()0
`
`%Survival
`
`lthrlddllnC, O
`
`a ThiOTidaZinC.
`
`lug/ml
`
`ThiOFidflZine, Eng/ml
`
`\\\\\\\x\\x\\
`
`Thioridazinc, O
`
`E21
`
`Thioriduzine, lug/ml
`
`Thioridazine, 2pg/ml
`
`'\ix
`"5\\)\,
`
`xx\\\\\\.rxxxw'xfxl.’\\~\x\\\
`
`\.
`
`x
`
`xx\x
`
`.
`\.
`a.
`
`aK
`
`_\a.
`\
`
`\\\\\\\\\\\\\\\\\\\\
`
`III/IIIIJIIIIIIIII‘II.’
`
`\\\\\x\\\\\\\\\\\\\\
`
`{III/JIIJIIIIZIIIIIII
`
`
`
`
`0
`
`0.05
`TaxolQrM)
`
`0.1
`
`0
`
`0-05
`TaxolflilVI)
`
`0~1
`
`(a), (c) and (d). Thioridazine had no significant Taxol sensitization efTect either on the parental MDA-MB—435 cells or on
`Figure 4
`the p185-overexpressing 435.6B2 and 435.eB4 transfectants (b) Thioridazine had dramatic Taxol sensitization effect on the p170—
`overexpressing CHO.VBR1,5-l cells. The indicated cells (~2 X 104) in 0.1 ml culture medium were plated onto 96—well plates. 24 h
`later, cells were grown in either 100 ,ul fresh medium as drug-free control; or 100 ,Lll fresh medium containing Thioridazine (l or
`2 rig/ml); or 100 [11 fresh medium containing different concentrations of Taxol; or lOO ,ul fresh medium containing thioridazme plus
`Taxol. Cells were incubated for 72 h, and the percentage of cells surviving from each group were determined by usmg the
`CellTiter96TM AQ Non—Radioactive Cell Proliferation Assay (Promega Corp)._Taxol concentrations used in these assays were
`chosen to be close to the IC50 concentrations determined by using the CellTiter96rM AQ Non—Radioactive Cell Proliferation Assay,
`which were 0.05 or 0.1 itM for the breast cancer cells. 5 or 10 11M for the CHO cells
`
`
`
`

`

`
`1364
`
`Taxol resistance of p185-overexpressing breast cancer cells
`D Yu et al
`
`are likely to respond to certain chemotherapy regimens.
`A correlation between chemoresistancc and p185
`expression and cell proliferation characteristics has
`been reported in non-small cell lung cancer cell
`lines
`established from different patients (Tsai et al., 1996).
`However,
`the increased Taxol
`resistance in p185-
`overexpressing 435.eB breast cancer cells was clearly
`not accompanied by significant differences
`in cell
`proliferation characteristics, which suggests that over-
`expression of p185 in breast cancer cells may confer
`Taxol resistance via novel mechanisms. Increased drug
`resistance has been previously reported in NIH3T3
`cells transformed by the c-myc or ras oncogenes (Niimi
`er al., 1991), whereas our experiments demonstrated
`that
`increased Taxol
`resistance was
`an intrinsic
`
`property associated with c—erbB-Z/neu gene expression
`but not
`a phenotype
`associated with oncogenic
`transformation.
`
`Although the p185-overexpressing 435.eB transfec—
`tants demonstrated increased Taxol
`resistance com-
`pared with
`the MDA—MB-435,
`435.neo,
`and
`435.11eo.pool cells that expressed basal levels of p185,
`the levels of mdr-I—encoded P-glycoprotein p170 are
`similar among these cells.
`In addition,
`the Indr-I
`blocker
`thioridazine were not more
`effective on
`sensitizing the p185-overexpressing 435.eB transfec-
`tants
`to Taxol
`treatment
`than the MDA-MB-435
`
`parental cells. The cognate subtle increase (4710%)
`in Taxol sensitivity by the mdr-I blocker thioridazine in
`both cell
`lines are consistent with the similarly low
`p170 levels of these cell
`lines. These results clearly
`indicate that overexpression of c-erbB—Z/neu-encoded
`
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`expression or
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