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Keywords:

  • bladder carcinoma;
  • invasion;
  • chemosensitivity;
  • recurrence;
  • proline-directed protein kinase

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

It has been shown previously that proline-directed protein kinase FA (PDPK FA) is overexpressed in various human malignancies compared with its expression in normal controls, and the suppression of overexpressed PDPK FA is capable of inhibiting the growth of various types of human carcinoma cells, suggesting a role for this PDPK in human malignancies. In this report, the authors combine immunohistologic, molecular, cellular, and clinicopathologic studies to demonstrate further an essential critical role for overexpressed PDPK FA in bladder carcinoma invasion, chemoresistance, and poor prognosis.

METHODS

The expression and localization of PDPK FA were analyzed by the immunohistochemical staining of specimens obtained from patients with primary transitional cell carcinoma (TCC) of the bladder. The stable antisense clones of human bladder carcinoma cells with specific suppression of overexpressed PDPK FA were established for invasion and chemosensitivity studies.

RESULTS

The immunohistochemical study revealed that PDPK FA was overexpressed preferentially in the invasive bladder carcinoma tissues. It was found that the stable antisense clones with specific suppression of overexpressed PDPK FA to ≈40% of the parental control level were capable of inhibiting the invasive activity and simultaneously enhancing the chemosensitivity of bladder carcinoma cells to various therapeutic drugs, such as vinblastine, vincristine, paclitaxel, and bleomycin. Clinicopathologic studies also revealed a correlation between overexpressed PDPK FA and disease recurrence/survival in patients with primary TCC (P < 0.05).

CONCLUSIONS

Taken together, the results demonstrate an essential critical role of overexpressed PDPK FA in invasion, chemoresistance, and poor prognosis. Suppression of overexpressed PDPK FA may provide a new potential target for therapeutic intervention aimed at preventing chemoresistance, disease progression, and recurrence in patients with bladder carcinoma. Cancer 2002;95:775–83. © 2002 American Cancer Society.

DOI 10.1002/cncr.10731

Bladder carcinoma is the seventh most common malignancy and the eighth leading cause of cancer death in the world, and the number of new cases has increased annually over the past 20 years.1 About 70% or more of the patients who are diagnosed initially with superficial bladder carcinoma do not face a life-threatening situation; however, 50–75% of these patients will develop recurrent disease.2, 3 The recurrence rate or bladder carcinoma is unusually high despite the availability of transurethral resection, and progression of superficial bladder carcinoma to deep invasion will occur. Although chemotherapy is capable of prolonging the interval to recurrence, unfortunately, no chemotherapeutic drugs have been found to reduce disease progression and metastasis.4–7 The challenges of controlling bladder carcinoma are the prevention of recurrent disease and the inhibition of disease progression during the treatment course.

It has been shown previously that proline-directed protein kinase FA (PDPK FA) is overexpressed in various human malignancies compared with its expression in normal controls,8–11 and the suppression of overexpressed PDPK FA is capable of inhibiting the malignant growth of various types of human leukemia cells12, 13 and of enhancing chemosensitivity in human prostate carcinoma cells14 and acute lymphoblastic leukemia cells,15 suggesting a role for this PDPK in human malignancies. In this study, first, we used immunohistochemical studies to demonstrate further that PDPK FA is overexpressed preferentially in invasive bladder carcinoma tissues. To confirm the functional role of this overexpressed PDPK in bladder carcinoma, two stable antisense clones of human bladder carcinoma cells (T24) that express ≈40% of PDPK FA existing in parental cells were selected and characterized. The results indicated that specific suppression of overexpressed PDPK FA in human bladder carcinoma cells is capable of inhibiting the invasive activity and simultaneously potentiating chemosensitivity toward various therapeutic drugs, such as vinblastine, vincristine, paclitaxel, and bleomycin. Clinicopathologic studies further confirmed a correlation between the expression of PDPK FA and disease recurrence and survival of patients with primary transitional cell carcinoma (TCC) of the bladder. The current report establishes the inhibition of overexpressed PDPK FA as a potential target for preventing chemoresistance, disease progression, and recurrence of human bladder carcinoma during and after treatment.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patients

We examined 63 patients with primary TCC of the bladder (48 male patients and 15 female patients) who underwent transurethral resection at National Taiwan University Hospital, Taipei, Taiwan during the period from January, 1994 to September, 1995. The average age of patients at the time of surgery was 60.3 years (range, 27–78 years). All available primary tumor tissues were fixed in buffered formalin solution, embedded in paraffin, and stained with hematoxylin and eosin for histologic evaluation. The clinicopathologic stage of the tumors was classified according to the Marshall modification of the Jewett–Strong system.16 The histologic degree of differentiation was graded according to the World Health Organization classification system.17 None of the patients received irradiation or chemotherapy before surgery. The follow-up was > 5 years until death or the end of August, 2001.

Immunohistochemical Staining

Tissue sections (5 μm) were sliced using a microtome, mounted on polylysine-coated clean glass slides, deparaffinized in xylene, and rehydrated in graded concentrations of ethanol. Endogenous peroxidase was inhibited with 3% hydrogen peroxide followed by blocking with 1 mg/mL of bovine serum albumin for 5 minutes. Next, slides were incubated with anti-PDPK FA antibody (2 μg/mL) at 4 °C for 18 hours and then with biotinylated antirabbit immunoglobulin G antibody for 10 minutes, followed with peroxidase-conjugated streptavidin label (LSAB® 2 system; DAKO, Carpinteria, CA). Color was developed using 3-3′-diaminobenzidinetetra hydrochloride. Hematoxylin was used to counterstain the slides.

Scoring of Immunohistochemical Staining

The extent of PDPK FA immunoreactivity was classified ultimately as PDPK FA negative and PDPK FA positive. The PDPK FA negative group consisted of those tumor cells with no detectable (−) or only very low levels (+) of PDPK FA immunoreactivity. The PDPK FA positive group consisted of those tumor cells with moderate levels (++) or high levels (+++) of PDPK FA immunoreactivity. The highest staining intensity associated with bladder carcinoma was used as the major determining parameter. Scoring was determined by two independent observers.

Cell Culture and Selection of PDPK FA Antisense-Expressing Clones

Human bladder carcinoma (T24) cells (Grade 3 TCC of the bladder18; American Type Culture Collection, Rockville, MD) were transfected by PDPK FA antisense expression vector and were cultured selectively under conditions essentially similar to those described in a previous report.14 The bladder carcinoma cells within passages 5–20 were used for all of the experiments described in this text. The detailed procedures for the production and characterization of anti-PDPK FA antibody, for immunoblot analysis of PDPK FA in crude cell extracts, for cloning of PDPK FA cDNA and construction of recombinant antisense expression vector, and for transfection and characterization of the stable transfected bladder carcinoma cells with specific suppression of PDPK FA expression were essentially as described in previous reports.12–15, 19

Tumor Cell Invasion Assay

Tumor cell invasion assay was performed using a modified Boyden chamber essentially as described by Albini et al.20 Briefly, a polycarbonate filter (Millipore, Bedford, MA) with 8-μm pores coated with 12.5 μg of Matrigel (Becton Dickinson, Heidelberg, Germany) was used as a barrier. The lower compartment was filled with culture medium containing 10% fetal bovine serum and 25 μg of fibronectin (Sigma, St. Louis, MO). The cells (2 × 105) were placed in the upper compartment of the chamber in serum free medium and incubated at 37 °C. After a 24-hour incubation, the cells that migrated to the lower compartment were fixed, stained with hematoxylin and eosin, and counted. The invasive activity was expressed as the number of migrated cells per chamber.

Drug Sensitivity Assay

Drug sensitivity assay was determined by staining the viable cells with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) solution (MTT assay), as described by Hansen et al.21 Briefly, bladder carcinoma cells (5 × 103) cultured in 96-well microtiter plates with different concentrations of various drugs at 37 °C for various time intervals were stained with 1 mg/mL MTT solution for 4 hour. Colored formazan converted by viable cells from tetrazolium salt was resolved in 100 μL of dimethyl sulfoxyde at room temperature for 15 minutes. Absorbance was read at 570 nm using an enzyme-linked immunosorbent assay reader. The viable cells, expressed as the percentage of untreated cells, were determined.

Statistical Analysis

For a correlation analysis between various parameters, chi-square tests or Student t tests were used for dichotomized values. For comparisons between two parameters, P < 0.05 was considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Preferential Overexpression of PDPK FA in Invasive Bladder Carcinoma Tissues

The protein expression and localization of PDPK FA were analyzed first by immunohistochemical staining of the human bladder carcinoma tissues. Figure 1A shows a typical, representative PDPK FA staining pattern with strong immunoreactivity of PDPK FA in tissues from a patient with primary TCC of the bladder compared with the adjacent normal tissues, which are negative for PDPK FA staining. It is interesting to note that PDPK FA was overexpressed preferentially, particularly in the obviously invasive TCC tissues (Fig. 1B). Similar staining patterns were observed consistently in 42 of 63 carcinoma tissue samples (66.7%) that were obtained from patients with primary TCC of the bladder (data not shown). This is the first indication of an association of PDPK FA overexpression with human bladder carcinoma invasion.

thumbnail image

Figure 1. Representative immunohistochemical staining patterns of overexpressed proline-directed protein kinase FA (PDPK FA) in tissues from patients with bladder carcinoma. The bladder carcinoma tissues obtained from patients with primary transitional cell carcinoma (TCC) were subjected to immunohistochemical staining using anti-PDPK FA antibody (2 μg/mL), essentially as described in the text (see Materials and Methods). The typical, representative staining patterns in 42 tissue samples showed strong immunoreactivity for PDPK FA in patients with primary TCC (A) and even stronger immunoreactivity for PDPK FA in patients with invasive TCC (B) compared with the findings in adjacent normal tissues, which were negative for PDPK FA staining (original magnification, ×100 A, B).

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Suppression of Overexpressed PDPK FA Inhibits the Invasive Potential of Human Bladder Carcinoma Cells

To further confirm the role of overexpressed PDPK FA in human bladder carcinoma invasion, the antisense clones of human bladder carcinoma cells (T24) with specific suppression of PDPK FA were selected and characterized, as described above (see Materials and Methods), and then were subjected to the invasion study. Although the doubling time of the antisense clone with suppressed PDPK FA to ≈37% of the parental control level was prolonged only from 22.2 hours to 28.8 hours, the invasive activity of the antisense clone was inhibited dramatically compared with the parental clone, as evidenced by the migrated cell numbers in the invasion chamber. More than 90% of the invasive potential of the bladder carcinoma cells could be blocked when the PDPK FA level was suppressed to ≈37% of the control level. All of these results are summarized in Table 1. Taken together with the histologic results showing the preferential overexpression of PDPK FA, particularly in the invasive bladder carcinoma tissues, the results are in consistent with the notion that the overexpression of PDPK FA indeed may play an important role in regulating human bladder carcinoma invasion.

Table 1. Biologic Properties of Proline-Directed Protein kinase FA Antisense Clones of Human Bladder Carcinoma Cells (T24)
Cell cloneaProtein level of PDPK FA (% of control)bDoubling time (hours)cInvasive activity (migrated cells per chamber)dInvasive potential (% of control)e
  • PDPK FA: proline-directed protein kinase.

  • a

    W.T., parental cells; As1 and As2, the PDPK FA antisense clones of human bladder carcinoma cells (T24).

  • b

    Determined by immunoblot analysis.

  • c

    Calculated during the exponential growth phase.

  • d

    Cells (2 × 105) in the serum free medium in the upper invasion chamber were incubated at 37 °C for 24 hours. The migrated cells to the lower chamber were fixed, stained with hematoxylin and eosin, and counted. The invasive activity was expressed as the number of the migrated cells per chamber.

  • e

    Represented as the percentage of the migrated cells per chamber. All data were taken from the averages of at least three independent experiments and are expressed the mean ± standard deviation, and the values are statistically significant with P < 0.05 (Student t test).

W.T.10022.2 ± 3.3857 ± 65100
As141.0 ± 4.727.7 ± 0.592 ± 7.110.8 ± 1.6
As237.2 ± 4.728.8 ± 1.376 ± 8.58.9 ± 1.7

Suppression of Overexpressed PDPK FA Potentiates the Chemosensitivity of Human Bladder Carcinoma Cells

In addition to the loss of invasive potential, the antisense clones described above also displayed enhanced chemosensitivity to various anticancer drugs. Figure 2A shows that the antisense clones with suppressed PDPK FA potentially enhanced the chemosensitivity of vinblastine in human bladder carcinoma cells. It is important to note that, when the concentration of vinblastine increased from 0.01 nM to 1 nM, parental bladder carcinoma cells remained highly resistant to the drug, with ≈80% viability. In contrast, the viable cells of both antisense clones significantly decreased to ≈30% of the control level (P < 0.05). Similar results with enhanced chemosensitivity in the antisense clones of human bladder carcinoma cells with suppressed PDPK FA also were obtained when vincristine (Fig. 2B), paclitaxel (Fig. 2C), and bleomycin (Fig. 2D) were used. The 50% inhibitory concentration (IC50) index, as summarized in Table 2, revealed further that the antisense clones with low levels of PDPK FA displayed ≈780-fold sensitivity toward vinblastine, ≈420-fold sensitivity toward vincristine, ≈140-fold sensitivity toward paclitaxel, and ≈10-fold sensitivity toward bleomycin compared with the parental bladder carcinoma cells. All of the results taken together demonstrate that the suppression of PDPK FA in bladder carcinoma cells is capable of inhibiting the invasive activity and simultaneously potentiating chemosensitivity in response to various therapeutic drugs. The relative sensitivities of the parental and antisense clones to clinically relevant concentrations of paclitaxel (10 nM) (Fig. 3A) and vinblastine (3 nM) (Fig. 3B) were tested, and the isodose sensitization enhancement also could be observed, supporting the clinical implications of the current study.

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Figure 2. The suppression of overexpressed proline-directed protein kinase FA (PDPK FA) enhanced various therapeutic drug sensitivities in human bladder carcinoma cells. PDPK FA antisense clones As1 (squares) and As2 (triangles) and parental T24 cells (circles) at a density of 5 × 103 per well in 96-well plates were incubated with various concentrations of therapeutic drugs, as indicated, at 37 °C (vinblastine, vincristine, and paclitaxel for 48 hours; bleomycin for 24 hours). Data were taken from the average of three independent experiments and are expressed as the means ± standard deviation (error bars).

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thumbnail image

Figure 3. Isodose sensitization enhancement in human bladder carcinoma cells to clinically relevant concentrations of therapeutic drugs. Proline-directed protein kinase FA (PDPK FA) antisense clones As1 (squares) and As2 (triangles) and parental T24 cells (circles) at a density of 2 × 104 per well in 24-well plate were incubated with 10 nM paclitaxel (A) or 3 nM vinblastine (B) at 37 °C for various time intervals, as indicated. Data were taken from the average of three independent experiments and are expressed as means ± standard deviation (error bars).

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Table 2. Enhanced Drug Sensitivity in Proline-Derived Protein Kinase FA Antisense Clones of Human Bladder Carcinoma Cells (T24)a
Cell cloneVinblastineVincristinePaclitaxelBleomycin
IC50 (nM)FoldIC50 (nM)FoldIC50 (nM)FoldIC50 (μM)Fold
  • a

    The drug sensitivity in terms of 50% inhibitory concentration (IC50) was determined by cell viability analysis (3-[4,5-dimethylthiazol-2-yl]-diphenyl-tetrazolium bromide assay) of parental (W.T.) and antisense (As1 and As2) clones of human bladder carcinoma cells (T24). All the cell clones (5 × 103) in different concentrations of drugs, as indicated, were incubated at 37 °C for 48 hours (vinblastine, vincristine, and paclitaxel) or for 24 hours (bleomycin). Data were taken from the averages of at least three independent experiments, and the values are statistically significant with P < 0.05 (Student t test).

W.T.64.21.045101.0476.41.031.21.0
As10.093690.314.7306.85.783.63.58.9
As20.082782.910.8417.63.4140.13.110.1

Correlation between Overexpressed PDPK FA and Prognosis for Patients with Primary TCC of the Bladder

Because overexpressed PDPK FA may play an essential role in regulating multidrug resistance and invasion/progression of human bladder carcinoma, and because poor chemosensitivity and unexpected invasion/progression are the two key factors in determining the poor prognosis of patients with primary TCC of the bladder,2–7 it is highly possible that overexpressed PDPK FA may be associated with the clinicopathologic outcome of patients with bladder carcinoma. To confirm this point, clinicopathologic studies were performed. Table 3 shows that 41 of 42 patients (> 97%) of patients who had primary TCC of the bladder with positive PDPK FA expression developed either local recurrence or distant metastasis during the 5-year follow-up study. In sharp contrast, only 4 of 21 patients (< 20%) who had primary TCC of the bladder with negative PDPK FA expression developed local recurrence but did not develop distant metastasis under the same follow-up conditions. It is even more noteworthy that 15 of 42 patients (≈ 35.7%) who had positive PDPK FA expression died within 5-year follow-up; whereas, under the same conditions, none of the patients with negative PDPK FA expression died during the same period, as shown in Table 3. Statistical analysis revealed further that, although there were no significant correlations of the expression of PDPK FA with age, gender, or histologic grade, there was a statistical correlation between overexpressed PDPK FA and recurrence/survival of patients with primary TCC of the bladder (P < 0.05) (see Table 4).

Table 3. Comparisons of the Expression of Proline-Directed Protein Kinase FA with the Clinicopathologic Outcomes of Patients with Primary Transitional Cell Carcinoma of the Bladdera
PatientPDPK FAAge (yrs)GenderGradeLocal recurrenceMetastasisDeath
  • PDPK FA: proline-directed protein kinase FA; M: male; F: female.

  • a

    Grade of PDPK FA expression was defined as positive (≥ 2+ intensity) and negative (≤ 1+ intensity), as described in Materials and Methods. Recurrence, metastasis, and death all occurred within 5-year follow-up. The cause of deaths was from bladder carcinoma.

1Positive27M2+++
2Positive58M2+++
3Positive72M2+++
4Positive58M2+++
5Positive75F2+++
6Positive64M3+++
7Positive54M3+++
8Positive72F3++
9Positive66M2++
10Positive62M1++
11Positive71M3++
12Positive62M2++
13Positive53M2++
14Positive56M2++
15Positive61F2++
16Positive75M2++
17Positive52M2++
18Positive43M2++
19Positive58F3++
20Positive66M2+
21Positive49M1+
22Positive72M2+
23Positive74F2+
24Positive57M2+
25Positive74F2+
26Positive32M2+
27Positive49M1+
28Positive45M2+
29Positive68M2+
30Positive64M2+
31Positive46M1+
32Positive69F3+
33Positive67M1+
34Positive71M1+
35Positive78F1+
36Positive75M2+
37Positive52M2+
38Positive56M2+
39Positive63M1+
40Positive47M2+
41Positive64M3+
42Positive68M2
43Negative53F3
44Negative71M3
45Negative55M2
46Negative67M1
47Negative43M2
48Negative58M2
49Negative65F3
50Negative73M1
51Negative76M2
52Negative34M2
53Negative70M3
54Negative64F2
55Negative59F2
56Negative48F2
57Negative61F1
58Negative58M1
59Negative49M3
60Negative63M2+
61Negative46M2+
62Negative70F3+
63Negative70M3+
Table 4. Statistical Correlation of the Expression of Proline-Directed Protein Kinase FA with the Clinicopathologic Outcomes of Patients with Primary Transitional Cell Carcinoma of the Bladder
CharacteristicNo. of patientsNegative (n = 21 patients; 33.3%)aPositive (n = 42 patients; 66.7%)aP valueb
  • SD: standard deviation; NS: not statistically Significant.

  • a

    The grade of proline-directed protein kinase FA expression was defined as positive (≥ 2+ intensity) and negative (≤ 1+ intensity), as described in Materials and Methods.

  • b

    Chi-square or Student t test was used.

  • c

    Recurrence covered local recurrence and distant metastasis and occurred within the 5-year follow-up period.

  • d

    The patients that died mainly due to recurrence and metastasis were selected to calculate the survival probability.

Age in yrs (mean ± SD)59.7 ± 11.160.6 ± 11.80.7649 (NS)
Gender    
 Male4814340.2095 (NS)
 Female1578
Histologic grade    
 112480.2994 (NS)
 2371027
 31477
Recurrencec    
 Yes45441< 0.0001
 No18171
Five-year survivald    
 Yes4821270.001704
 No15015

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

In this study, first, we used immunohistochemical staining to demonstrate that the multisubstrate/multifunctional PDPK FA is overexpressed in human bladder carcinoma tissues relative to normal control tissues and is overexpressed preferentially, particularly in the invasive areas, suggesting an association of overexpressed PDPK FA with human bladder carcinoma invasion. Subsequently, we used a direct cell-engineering approach to demonstrate further that specific suppression of overexpressed PDPK FA is able to inhibit the invasive potential of human bladder carcinoma cells, confirming the immunohistochemical results that PDPK FA indeed may play an important role in regulating the invasive activity of human bladder carcinoma cells (see Table 1). The final clinicopathologic study also confirmed a close correlation of overexpressed PDPK FA with recurrence and survival in patients with primary TCC of the bladder (see Table 4). This is the first report providing initial evidence to demonstrate an essential critical role of overexpressed PDPK FA in disease progression and prognosis for patients with bladder carcinoma.

It is believed generally that poor chemosensitivity and unexpected invasion/progression are the two key factors in determining recurrence and poor prognosis in patients with primary TCC of the bladder.2–7 Although intravesical chemotherapy after transurethral resection may result in a short-term decrease in the bladder carcinoma recurrence rate, there is no clear advantage of such chemotherapy with respect to bladder carcinoma invasion, and the prognosis of patients with invasive bladder carcinoma remains poor despite the improved surgical technique and combined chemotherapy.4–7 Because there is no effective treatment for preventing bladder carcinoma invasion, it remains difficult to improve the survival rate for patients with bladder carcinoma.2–7 In view of the facts presented here showing that the suppression of overexpressed PDPK FA is capable of inhibiting the invasive activity and simultaneously enhancing the chemosensitivity of bladder carcinoma cells, it is highly possible that the combination of enhanced chemosensitivity with inhibition of invasion through the suppression of overexpressed PDPK FA synergistically may promote more complete remission for patients who have primary TCC of the bladder with positive PDPK FA expression.

Consistent with this notion, clinicopathologic studies also have revealed a correlation between overexpressed PDPK FA and recurrence or survival in patients with bladder carcinoma who have primary TCC. Taken together with all of the molecular, cellular, and clinicopathologic results presented here, the current study establishes the inhibition of PDPK FA as a new potential target for therapeutic intervention aimed at enhancing chemosensitivity and simultaneously inhibiting invasion to prevent recurrent bladder carcinoma in patients who have primary TCC with positive PDPK FA expression.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES